MOLECULAR BIOLOGY o N95 respirator mask, goggles or faceshiels LABORATORY WORKFLOW

Velez College: Batch Celestia (S.Y. 2021-2022) • Disinfect all working areas, surfaces, and equipment
Note: these are UNOFFICIAL NOTES. Always make it a habit before and after use with 10% bleach followed by
to refer to the lecture video and clarify information with 70% alcohol
the suggested reference books or lecturer.
LECTURE/LAB TOPIC: PCR Part 3 – Real-Time Assays & LABORATORY LAYOUT
Troubleshooting
LECTURER: Nina Christine O. Orilla, RMT, MLS (ASCPi)

INTRODUCTION

In this lecture we will be discussing the buffer preparation

until the data analysis.

REAL-TIME PCR PRINCIPLES

To understand it more clearly, your PCR or also your target
amplification is analogous to growing cells in culture and
*Good to know: A unidirectional workflow must be
allowing the cells to replicate thus nucleic acids as well as
observed; working from a clean area to a dirty area in
themselves (?) so that it can be visualized in an agar plate.
order to prevent cross contamination.
However, waiting for cells to replicate to detectable levels
Picture shows how your Real time PCR Analysis graph Here, we consider the reagent preparation area as the can take days to weeks or months. Whereas, replicating
would appear that represents different dyes which in turn, clean area. In the dirty area is your amplification room, the nucleic acids in vitro (just like your real-time PCR) only
represents different target genes. Color-coded for us to your extraction room, etc. takes minutes to hours. Real-time PCR is the in vitro growth
easily recognize the dyes and the genes they represent. of nucleic acids.

Sample laboratory layout:

OBJECTIVES In the previous lectures, we learn that there are simplex
• Discuss the principles of qualitative rRT-PCR and multiplex assays.
• Follow the procedures in a new assay design (e.g.
new IFU) Multiplex PCR – there are more than one primer pairs that
• Summarize the procedures in 2019-nCov rRT-PCCR can be added to your PCR mix. Useful to detect not only
using Sansure Biotech Kit target genes but allows to confirm accurate detection of
• Explain what a Ct value is a single target
• Interpret the results and validity of controls
• Interpret the results of unknown RNAs PCR – Polymerase Chain Reaction
• Identify the laboratory rooms for each major steps - Process for the amplification of specific
• Explain troubleshooting in rRT-PCR assays fragments of DNA
Real-Time PCR
*In room 2, we also have aliquoting since we are a A specialized technique that allows a PCR
TOPIC OUTLINE -
reference laboratory, we send different samples to reaction to be visualized “in real time” reaction
• Introduction video: VSMMC SNL Video
different institutions for testing. The number of personnel progresses
• Real Time PCR Principles
would actually depend on the sample load that your - Allows estimation and measurement of minute
• Real Time PCR: SARS-COV-2 (2019-nCoV)
laboratory will be receiving. amount of DNA sequences in a sample
• Troubleshooting
• Learning Enhancements and Extensions - In real-time PCR, the use of probes allows the
amplification to be monitored in real time and
BIOSAFETY REMINDERS the DNA copies can be quantified.
• Wear Proper PPE REAL-TIME PCR APPLICATIONS
o Lab gown over scrub suit, disposable Real-Time PCR has become a cornerstone of molecular
waterproof lab gown, closed shoes with biology. Some of its uses include:
shoe covers

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 Lysis Buffer = Phenol and Guanidine Isothocyante

1. Disease diagnosis and management RT-PCR STEPS

a. Qualitative detection FIRST STEP: REVERSE TRANSCRIPTION
b. Viral quantitation
2. Gene expression analysis
a. Cancer and Drug research
3. Food testing
a. Percent GMO food
4. Animal and Plant breeding
a. Gene copy number
5. Forensics
a. Sample identification and quantification

Real-Time PCR in Disease Management:

Drug treatment for HIV infection often depends on
The first-strand complementary DNA synthesis is
monitoring the viral load. Real-time PCR allows for direct After the extraction of the viral RNA, the next step is the primed with the PCR reverse primer which hybridizes
measurement of the amount of the virus RNA in the preparation of the reaction mixture for PCR amplification. to a complementary part of the virus RNA genome.
patient. Reverse transcriptase then adds DNA nucleotides
HIV – detection of disease prognosis and efficacy of anti- In this step, a master mix is used which is a premixed onto the 3-prime
retroviral therapy concentrated solution, that consists of buffer, end of the primer, synthesizing DNA complementary
1 Reverse Transcriptase enzyme, nucleotides, of the viral RNA. The temperature and duration of this
REVERSE TRANSCRIPTION Forward Primer, Reverse Primer, TaqMan probe, step depend on the primer, the target RNA, and the
and DNA polymerase. reverse transcriptase used. (10 MINS)
Finally, to complete this reaction mixture, the RNA
2
template is added. SECOND STEP: INITIAL DENATURATION
The tube is Mixed by pulse-vortexing, then the
reaction mixture is loaded into a PCR plate, which
3
generally contain 96 wells allowing the analysis of
The viral RNA isolated from patient samples is reverse several samples at the same time.
transcribed (converted) into its “complementary” DNA 4 Next, the plate is placed in a PCR machine, which
(cDNA) to ensure that the genetic material is stable and is essentially a thermal cycler.
can be replicated during the PCR step.
Real-time RT-PCR is used for the detection of the new
Since RNA is single-stranded, easily degraded, and coronavirus 2019 by the amplification of target sequences
This step causes denaturation of the RNA-DNA
unstable, you need to stabilize it for the PCR steps. in the RdRP gene, the E gene, and the N gene. The choice
hybrids. This step is required for the activation of DNA
Because PCR steps include a lot of denaturation and of the target gene depends on the primers and the probe polymerase and
heating in different temperatures and in order to withstand sequences. simultaneously the inactivation of reverse
these conditions, we need a stable genetic material (DNA transcriptase.
is more stable). PCR consists of a series of thermal cycles, with each cycle
consisting of DENATURATION, ANNEALING, and EXTENSION
REAL-TIME PCR PRINCIPLES, INSTRUMENTS, PROCEDURES steps.

VIDEO: REAL-TIME PCR REACTION MIXTURE PREPARATION

TO DATA ANALYSIS
Link: https://www.youtube.com/watch?v=ThG_02miq-4
(starting at timestamp 3:25)

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 CYCLE 1 ▪ the denaturation of CYCLE 3
this double-stranded
STEP DESCRIPTION TEMP TIME DNA is performed, STEP DESCRIPTION TEMP TIME
DENATURATION 95oC 15s
yielding two single- With each cycle of
stranded DNA DENATURATION PCR, more dye 95oC 15s
▪ consists of
molecules molecules are
heating the
reaction chamber ▪ the reaction released, resulting in
temperature is an increase in 58
to 95°C ANNEALING oC
15s
DENATURATION ▪ used for 95oC 15s lowered, allowing fluorescence
denaturation of annealing of the intensity proportional
the double- primers to each of to 58
the single-stranded EXTENSION the amount of 15s
stranded DNA oC

template DNA templates and amplicon synthesized

annealing of the
▪ allowing
TaqMan probe to its
annealing of the This method allows the estimation of the amount of a given
complementary part
forward primer to sequence present in a sample.
of the target DNA
its The number of double-stranded DNA pieces is doubled in
complementary each cycle; therefore, PCR can be used to analyze
TaqMan Probe
part of the single- extremely small amounts of sample.
▪ consists of a
ANNEALING stranded DNA 58 oC 15s Detection
fluorophore
template
covalently attached (Measurement of the fluorescent signal)
▪ annealing
ANNEALING to the 5' end of the 58 oC 15s - a tungsten-halogen lamp, an excitation filter,
temperature relies
oligonucleotide mirrors, lens, an emission filter, and a charge-
directly on length
probe coupled device (CCD) camera are used.
and composition
▪ the fluorescence is 1. Filtered light from the lamp is reflected off mirror,
of the primers
emitted by the passes through a condensing lens, and is focused into
▪ the DNA
fluorophore when is the center of each well.
polymerase
excited by the 2. Then, Then, fluorescent light emitted from the wells
synthesizes a new
cycler’s light source
DNA strand reflects off the mirror, passes through an emission filter
▪ this probe also
complementary and is detected by the CCD camera.
consists of a
to the DNA In each PCR cycle, light from excited fluorophore can be
quencher at the 3'
template strand detected by the CCD camera which converts the light
end
by adding free that it captures into digital data. This method is known as
▪ the close proximity
nucleotides from REAL TIME PCR, which allows the monitoring of the progress
of the reporter to the
EXTENSION the reaction 58 oC 15s of the PCR reaction as it occurs in real time.
quencher prevents
mixture that are
detection of its
complementary
fluorescence In Real Time PCR, you will be able to check or monitor the
to the template in
▪ DNA polymerase progress in “real time”; you can see the growth or progress
the 5' to 3'
synthesizes new of the graph or curve as you go through time. Example:
direction
strands
▪ temperature at
▪ when the
this step depends
polymerase reaches
on the DNA
EXTENSION a TaqMan probe, its 58 oC 15s
polymerase used
endogenous 5'
nuclease activity
CYCLE 2
cleaves the probe,
separating the dye
STEP DESCRIPTION TEMP TIME from the quencher
*naka Bisaya si miss pag explain and much better jud I
watch nalang ehe*

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 - It also indicates an estimate on how much virus is
y-axis: estimated number of copies generated; in the sample to start with. Take note, estimate;
x-axis: number of cycles not the actual amount.
We can easily see that the left-right shift in the curves is
related to the starting quantity of DNA
In cycle 26, there could already be 2,000,000 amplicons
and this is because of the doubling effect of the PCR. In
Ct (*cycle threshold*) values identify the curve positions,
cycle 27, there could already be 4,000,000 amplicons.
based on where they cross a threshold.
DNA Quantity and Ct values are related as:
Quantity ~ 2 ct
*watch 18:58 onwards to see miss explain*
The blue (left graph) and magenta line (right graph) is the
threshold line; once it crosses that blue line, it will be able
to detect the virus already.
- The exhaustion of the reaction components and
competition between the PCR products and
primers during the annealing steps, slow the PCR
product accumulation after the exponential
phase of growth until it finally plateaus.
- In every manufacturer kit for PCR, there are
always limits of detection since we are
performing in vitro testing; we will be the one to
enter the amount of primers present in the PCR
mix including the enzymes, the volume of probes,
etc.
- Realistically, as the chain reaction progresses, it
gets exponentially harder to find primers, and
nucleotides. And the polymerase is wearing out.
- So, exponential growth does not go on forever.
If we plot the amount of DNA in our tube going forward
from cycle 25, we see that it actually looks like this.
(reaches a plateau phase)

Good to know: the threshold lines can be adjusted The fluorescent signal detects the growing target copy
(Another example) If there are 50,000 copies made at
manually or automatically. If done manually, you have to during the amplification process; analysis is performed in
cycle 30, it will double to 100,000 in the next cycle, cycle
base it upon the protocol that you are using. the exponential phase of growth.
31.

As you can see, the curves resemble the bacterial growth Because the length of the lag phase is inversely
curve. There are different phases as well (lag phase, proportional to the amount of starting template,
exponential growth phase, a linear phase, and a fluorescence will reach exponential growth in early cycles
stationary phase). when a lot of targets are present.
(Some doctors will ask for the Ct values to see if their
patients are infectious still; however, we don’t release Ct
values because this is just an estimate of the amount of
virus in the sample; these are not actual values). But for this
As PCR cycle progresses, the fluorescent signal intensity lecture’s purpose, the lower the Ct values are, the larger
increases. This signal can be detected and monitored by the amount of viral RNA/target gene present in the
(example of the PCR’s doubling effect) Let’s compare. sample. It has an inversely proportional relationship. For
the real-time PCR machine.
What if you start with four times as much DNA as tube 1? example, the Ct value is 23, it would be likely for a specific
Let’s picture two tubes at cycle 25 and work backwards a protocol which may have a cutoff of 30 (Ct value) that
x-axis: number of cycles
few cycles. would be a low Ct value indicating that the patient
y-axis/delta: delta rm (?), target genes/ number of copies
At cycle 23, Tube 1 has already made 250,000 amplicons probably has a larger amount of viral DNA/target gene in
of genes
while Tube 2 has 1,000,000 amplicons – it has 4 times as the sample.
32, 36, 27 – Ct values/ cycle threshold value
much DNA. As expected, by the doubling effect of PCR,
you have roughly around 1,000,000 amplicons for Tube 1 When less target is present, fluorescence will not reach the
Why are Ct values important in real-time PCR testing?
and for Tube 2, you have 4,000,000 amplicons. exponential growth phase until later cycles.
- These values (32, 36, 37, for example) is the
actual number of cycles it takes for the PCR
Question: Will this go on forever?
machine/test to detect the virus in the sample.

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 REAL-TIME PCR: Sars-CoV-2 (2019-nCov)
Most of the samples that test as strong positives all were
found after low number of cycles. We consider 16 cycles,
9, or 10 cycles to be really strong positive samples
especially if both target genes are present.
The different primers and probes used in different kits are
used to find and detect portions of SARS-CoV-2 specific
genes.
SARS-CoV-2 specific genes:
1. RdRp (RNA polymerase gene),
2. E gene (envelope gene),
3. N gene (Nucleocapsid) – nucleoprotein that
holds the genome (more sensitive and is more
superior than the ORF-I ab)
4. ORF-I ab,
5. Nsp
For our lab activity, we will base it on the Sansure kit since
it is one of the most commonly used detection kits used
here in the Cebu City. It uses the ORF-I ab and the N gene
as the target genes. There are also other detection kits that
use 3 genes: RdRp, E gene, and N gene. There is also
another kit that only uses one gene: ORF-I ab gene. The
genes used depend on the manufacturer.
REAL-TIME PCR: Reagent Preparation
In real-Time PCR, you will be preparing reagents such as
the Master Mix and buffers. Here, in master mix
preparation or reagent preparation, this is the set up
1. Cold Racks both for reagents and PCR plate or strip
2. Forceps
3. Marker pens
4. Tube cap opener
5. Micropipette
6. Pipette tips
7. PCR tubes • The number of negative controls you have your
Make sure that the laminar flow PCR cabinet has been positive control, in our Censure Biotech detection. ??
disinfected before and after use; as well as its contents. • Our assay worksheet, we incorporate 2 negative
Real-Time PCR: Reagent Preparation controls and 2 positive controls in one no template
control.
• As you can see there is a 1.1, it is a pipetting error.
• But the pipetting error would vary the institution
depending on how much pipetting error you would
incorporate.
• For example, you have number of total number of
samples
A little reminder for the reagent preparation in RT-PCR
• 22 samples multiplied by 1.1 = you will be preparing
• Always keep your reagents in a cold rack. To maintain
the master mix good for 22 samples
the cold chain?? of your sample to prevent
• Pipetting error will compensate the droplets, or the
degradation of the products.
fluids sticks into the pipette tips, and the PCR tubes.
• Since here you have your enzyme mix, PCR mix
(Primers, probes, TPS).
Real-Time PCR: Master Mix Preparation
• Take note that when for your enzyme mix that is
provided by the manufacturer, you only have to spin
it down. No need for pulse vortex for the enzyme.
Enzyme will be easily degraded and it is insensitive(?).
• For PCR mix (buffers, MTPS, primers and probes), pulse
vortex then spin down.
• Make sure that the reagents have been thawed
completely. (Do not force it to expose it to heat, just
let it stay in the cold rack, it will thaw through time)
• In opening the detection kit, you will have different
Real-Time PCR: Worksheet preparation components.
• 2 tubes, one for each component: Component A
(PCR mix) and Component B (PCR enzyme mix)
• This image is only for example of RT-PCR kit
component, later there will be a specific volume for
your Censure Biotech detection kit.
Real-Time PCR: Template Addition
• After reagent preparation, you will prepare your
worksheet.
• This is based on the Philippine Genome Center, a
protocol given to all national laboratories. This is how
the worksheet preparation would actually look like.
• Here you will determine many patient samples you will
be needing.
Depending on the institution, the setting we have full After creating the worksheet, and you’ll make the master
plates, 96 samples, we will accumulate all the wells in the mix of your PCR and you will dispense this master mix into
PCR plates. your PCR plate or PCR wells. Basing on your worksheet, the
• Five of which will be the controls and 91 will be the format of your worksheet is similar to the PCR plates. After
unknown samples. dispensing the master mix, you will dispense the template
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addition. The volume of the template would depend on until when it is excited by the cycles light Real-Time PCR: Probes
different protocols and deduction kits?. source.
It is used in the multiplex PCR testing.
Real-Time PCR: Cycling Condition It was developed as one of the first pro
based system for quantifying your
complementary DNA.
It measures the fluorescent signals
generated by the separation of fluorescent
dyes and quencher.
is present in the reaction mix in addition to
the specific primers that prime the DNA
Probe synthesis reaction Focus on the SansureTM Biotech 2019n-CoV Diagnostic
It is with primers, dNTPs, which is contain in Kit.
the PCR mix provided by the manufacturer
FAM ORF-1ab gene
• After template addition, you will load the PCR plate • Example of the simplex is the influenza, you will be the Specific Probe
ROX N gene
into the real time PCR machine or thermal cycler. one who’ll dispense a specified volume of your probe 1Human-
• You set up the protocols and thermal cycling to each master mix Cy5 RNase P
Specific Probe
conditions.
You have to incorporate it to know that the sample is
• Each time have a different temperature Real-Time PCR: Fluorophores collected properly and it is from a human being, this
requirements, different duration and cycles. why it is called an Internal Control.
Denaturation: 95C this step is required for the activation
of DNA polymerase simultaneously and inactivation of
Each probe uses a different fluorophore, so that the signal
your reverse transcriptase.
from each probe can be distinguished within a single
Annealing: lower temperature 59C, allows the
sample.
annealing of the forward primer to its complement part
of your single stranded DNA template Current SARS-CoV-2 RT PCR kits simultaneously detect one
Extension: your DNA polymerase synthesizes a new DNA to two viral genes and the human IAC gene. This is made
strand. possible with the use of different primers and fluorescent
Fluorophores adds color to the process.
• The Thermal Cycling condition in the image is not Different reporter dyes: FAM, HEX, Cy5, ROX, VIC probes included in the RT-PCR test kit.
constant and it would still depend on the protocol • • Every time you do the process, don’t forget to read
Each has corresponding colors for easier detection
you are using. through the package insert and instructions for use
and analysis. (You can change the colors, it is up to
just provided by the manufacturer.
your preference what color you want for the FAM and
Real-Time PCR: Detection As multiple copies of viral and human genes are made the
others)
fluorescence intensity of the different colored fluorophores
• Each reporter dyes has a corresponding target
increases. The fluorescent signal are simultaneously
genes.
detected by the real-time PCR machine using different
FAM reporter for ORF1ab gene
channels.
dye
Real-Time PCR: Instruments
ROX reporter dye for N gene
Since it is color coded, you can see in the that there is a
amplification, there is an S curve or sigmoidal curve that’s
color blue, so you know that the FAM
How do we detect the amplicons produced in your real reporter/fluorophores, there’s an amplification in ORF1ab
time RT-PCR? gene
• It uses probes to detect the real time progress of your • Fluorophores are like colors but just name differently.
copy generation of your DNA. (instead of red, pink blue, they are named as FAM,
One of the common probes is the Taqman ROX and others)
probes, it consists of fluorophores covalently • It is incorporated in different thermal cycler machine.
Taqman
attached to the 5’ prime and
probes Image: Thermal Cyclers available in the Subnational
oligonucleotide probe.? So the
Molecular Biology laboratory. BioRad thermal cycler – for
fluorescence is emitted by the fluorophore,
simplex assay influenza testing Sansure PCR thermal

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 cycler and ABI Applied biosystem thermal cycle. In total Real-Time PCR: Instruments Date and time specimen
Test results
we have 6 current thermal cyclers. received
Real-time PCR systems consist of THREE main Collection Interpretation
components: Result releasing
1 Thermal Cycler (PCR Machine), linked to a… • It is also the recommendation of the ___ to include
Optical Module (to detect fluorescence in the what test was performed.
2
tubes during the run), linked to a… (2nd image) In here, the test performed is the SARS-CoV2
Computer (to translate the fluorescence data into Viral RNA Detection Test (PCR)
meaningful results, that is why it is called real-time In tests result, you can release it as SARS-CoV 2 which is the
3
since you will able to see the real time progress of causative agent of COVID-19 viral RNA not detected or
The final product of real-time PCR is a table of Ct values
the PCR step) detected
from which amounts of DNA can be determined. What’s
important here is the Ct values and the interpretation of For the interpretation, either be negative or positive for
The computer, running real-time software, converts the SARS CoV-2 Viral RNA
the Medtech which would be validated by the pathologist
fluorescent signals in each well to meaningful data. • And there’s always a note below, that this laboratory
on duty.
• You can generate these results, print it out through results should be interpreted together with the
• They’re softwares that available clinical and epidemiological information
excel or word file pdf.
you input in the thermal You need to correlate and it is up to the clinician to
• You can see the different wells and what well it is
cyclers converts the correlate these results for his patients
specific of corresponding example ID, target names
fluorescent signal into • Negative for SARS CoV-2 Viral RNA result, there can
and dyes used.
meaningful data. To print be a false negative result that may arise due to the
For example, the detection of N gene and the dye used
the data, interpret and low level of virus in the sample due to poor sample
is the FAM dye, and what Ct values it has if there is an
validate the results. collection, handling, storage, timing of collection,
amplification on the target gene.
• In interpretation of the and presence of inhibitors.
• At 35 cycle, there’s an amplification, in this table
data, you can see the • That is why sometimes, the laboratory will be able to
(image above) you can see the results
different Ct values per target genes. release invalid results. Due to no RP or internal control.
Qualitative Detection of SARS-CoV-2 by rRT-PCR
(Sansure Biotech) o In Sansure kit, you have the human RNAse
WORKFLOW probe, to know you have collected properly
Laboratory Activity Basis
1 Set up PCR protocol from a human being
Depends on the SARS-CoV-2 detection kit o We will always go back to the sample
SARS-CoV-2 rRT-PCR Results
You will set the different thermal cycling conditions per collection, if you have collected the sample
step. (conditions on in the denaturation, its properly you will generate a good graph
temperature, time and what cycle) and good quality results
2 Set up plate layout 2019-nCoV Target Genes
How the 96 well plate format would
actually look like.
Plate layout is where you will input the
positions of your controls, and unknowns.
3 Collect data
4 Analyze data

After each run, you will have a

graph and have a different ORF1ab
(5 prime, big rectangle)
sigmoidal curve or S shaped gene
curves, threshold and Ct values. (3 prime end, square) Nucleocapsid
N genes
gene – where RNA genome is located
Both are inside the membrane and specific for the SARS-
This is how the rRT-PCR results would actually look like. And CoV-2 virus.
here are the details: This protocol is used for qualitative of the ORF1ab and N
Name of the patient Specimen type genes in nasopharyngeal, oropharyngeal swab, and
Patient identification Test performed tracheal aspirate samples.

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 2019-nCoV Nucleic Acid Diagnostic Kit • Reverse Transcriptase in RT mix Control for properly well in each sample
One cycle process: Denaturation, annealing and RNase P gene amplification
extension
Master Mix and Reporter Dyes
Kit Components of Sansure Biotech Table 1. Master mix prepararion
1 10 24 48
sample samples samples samples
Test principle: This test utilizes the 2019-nCoV Orf 1ab and
2019- 26 260 624 1248
the specific conserved sequence coding nucleocapsid
nCoV-PCR
protein N gene as the target regions
Mix (uL)
• Conserved in a way that the nucleocapsid/
2019- 4 40 96 192
nucleoprotein is in the 3-prime end nCoV-
• In every process, it is not always in exponential PCR-
growth, the components will be consumed and Enzyme
exhausted sooner or later Mix (uL)
Limit detection: 200 RNA copies per mL The sample 1 – Sample Release Reagent – used for the Note: The above configuration is for reference only
(Brown) PCR mix: where the probes, primers, extraction process (chemical extraction).
Left
enzymes and buffers are incorporated Buffer Master mix preparation: Always base on the IFU. For one sample, assuming it is a 26
to
(Transparent) Enzyme mix: Taq polymerase Sample 2 – 2019-nCoV-PCR-Mix – you have primers, uL of the PCR mix and 4 uL of Enzyme Mix. For 10 samples,
right
(White) Negative control probes, dNTPs, MgCl2 (acts as a cofactor for polymerase it would become 260 uL and 40 uL respectively (26 x 10, 4
tubes
(Pink) Positive control enzymes), Rnasin (a brand, protein inhibitor that inhibits x 10).
common contaminating RNases without destructing the
This protocol uses the positive internal control, which reverse transcriptase activity of the PCR), enzyme mix (2 For the lab activity, five unknown samples will be used and
monitors the presence of PCR inhibitors in test specimens enzymes: reverse transcriptase for reverse transcription three controls: NTC, Negative Control, and Positive
detecting whether the internal control signal is normal, to process and Taq polymerase enzyme (highly thermostable Control. So, a total of 8 plus 2 extra reactions since we must
avoid a false negative result. recombinant DNA polymerase, name after the thermos have a minimum of 10 reactions. If lower than 10, it will be
• There should always be an amplification, to know the aquaticus)), positive and negative controls (saline). affected by pipetting error.
internal control signal. thermos aquaticus: is a heat tolerant bacterium from GENERAL STEPS
which isolates itself 1. PCR Buffer or Master Mix Preparation
Components Why do we have to incorporate different controls? 2. RNA-template Addition
In a full plate set-up, in the Sansure protocol, we 3. Amplification
incorporate NTC that can be a negative control too. 4. Data Analysis
is used to monitor when there is
contamination in the extraction 1. PCR Buffer or Master Mix Preparation
Negative
process and used in each detection
control
run. It is provided by the
manufacturer.
to detect whether there is a
NTC
contamination during the buffer
(No template
Components of the protocol: PCR components which preparation on the template ▪ For one sample, mix 26 uL of PCR Mix, 4 uL of Enzyme
control)
have the probes, DNA sample, primers, nucleotides, dNTPs addition process. Mix with a total of 30 uL of Master Mix per sample. For
(deoxynucleotides triphosphate, they contain is used to monitor when there is 100 samples, 2600 uL of PCR Mix and 400 uL of Enzyme
deoxyribose which is the building block of DNA, 4 major contamination in the extraction Mix will be needed with a total of 3000 uL.
dNTPs which are commonly used such as DATP, DTTP, process ▪ Mix all of these in a PCR tube or Snap Cap Tube.
Positive control monitors whether the process works Note: 1-10 sample = add 1 extra reaction for pipetting
DGTP, DTTT) ▪
properly, probes, primers are error (This will vary per institution)
Nucleotides and dNTPs, help expand the growing DNA
functioning, therefore PC must have
strand with the help of the Taq polymerase (enzyme)
amplification in all genes.
Different probes: PCR tube, mix buffer, enzyme Taq
is used to monitor the sample
polymerase, sample (which is RNA extract), primers, and Negative/
collection, is the sample was
nucleotides. Internal
collected, handled and processed

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 2. RNA-template Addition ▪ FAM – for ORF1ab ▪ Negative Control (NC)
▪ ROX – for N gene ▪ Positive Control (PC)
▪ CY5 – human RNAse P gene or Internal Control ▪ No Template Control (NTC)
▪ Well A1 = NTC (no volume, no template added)
4. Data Analysis ▪ Dependent on the Lab instruction / SOP
▪ Make sure that controls are incorporated in every run
▪ Unknown = 20 uL + 30 uL = 50 uL

CONTROLS
▪ Proceed to RNA template Addition once the 30 uL 1. 2019-nCoV-PCR-Negative Control - a “no template”
mastermix is dispensed into the PCR plate (negative) control is used to monitor whether there is
▪ The RNA extract that was isolated during the contamination for the rRT-PCR process and is used in
extraction process will be used in this step. each detection run
▪ For Sansure Biotech, the extraction method can be ▪ At the end of the cycle/run, this is how it will look like. 2. 2019-nCoV-PCR-Positive Control - a positive template
used using qiagen extraction kit. LAB ACTIVITY: control is used to monitor whether the rRT-pCR
▪ The sample release reagent can also be used. process works properly and is used in each detection
▪ For Sansure Biotech Protocol, the Qiagen or sample run
release reagent can be used which is a chemical 3. An Internal Control for RNAse P gene - is used to
extraction method. monitor the sample collection, handling, and rRT-PCR
▪ Dispense 20 uL of extracted RNA (+ 30 uL of master process and is used in each sample amplification
mix). A total of 50 uL of PCR RNA Master Mix Mixture.
▪ Note: NTC or No Template Control = no template
added
o One way to determine whether there is
contamination along the process
o To identify the pipetting skills of technologists on
duty and the cleanliness of the process, as well.
▪ NTC is also considered as a Negative Control
▪ Targets: ORF1ab gene, N gene, RP (human RNAse P
3. Amplification
gene as IC/Internal Control)
▪ 1 Master Mix: 91 unknown + 5 controls = 96 reactions ▪ Fluoresence data is collected
Stage 1: 1 cycle 50 C for 30 ▪ A 96 plate reaction, dispensing 30 uL mastermix in ▪
Reverse Transcription minutes Reporter Dyes:
each well. o FAM – for ORF1ab
Stage 2: 1 cycle 95 C for 1
▪ In actual setting, incorporate 5 controls to make sure o ROX – for N gene
Initial Denaturation minute
that there is no contamination. o CY5 – human RNAse P gene or Internal
Stage 3: PCR 45
Control
amplification cycles 95 C for 15
▪ Denaturation: 95 C
▪ Denaturation seconds
▪ Annealing: Lowered to 60 C to allow the annealing of
▪ Annealing/Extension 60 C for 31
the forward primers to its complementary parts of the
seconds**
ssDNA
▪ The temperature relies on the length and composition
Stage 4: Cooling 1 cycle 25 C for 10
of the primer
seconds
**Fluoresence data is collected
Reporter dyes: FAM/ROX/CY5
▪ Denaturation: 95 C for 15 seconds; repeated every
after the end of the stage for 45 cycles
▪ Annealing/Extension: 60 C for 31 seconds; at this point
this is where Fluoresence data will be collected using
Reported dyes such as FAM/ROX/CY5

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 ▪ Most of the time, this kind of graph is obtained.
▪ Blue = background noise
▪ Pink = exponential PCR Phase; where the PCR started
making copies of the DNA
▪ Green = Threshold adjusted to fall within the PCR
exponential phase
▪ The threshold is above the background noise
because if you put the threshold below, it will read the
background noise.
Interpretation of Control Results
▪ The different thermal cycling conditions for Sansure
Biotech Protocol amplification
▪ Different protocols have different thermal cycling
conditions
▪ Before interpreting the individual unknown samples,
check first the control to know whether your run is
valid or not.
▪ First check the negative control. All NC, NTC, and
▪ After the run, the monitor will show the full plate / 96
extracted NC must have no CT value.
plate format.
▪ If there is CT value, it is greater than 40 which is
▪ For the activity, 8 samples will be incorporated
indicated in Sansure Biotech protocol.
▪ The graph will be interpreted
▪ Check the positive control for each target gene. It
▪ The different targets can be individually selected
must have a CT value of less than or equal to 35.
▪ The threshold can be set to Auto. For N gene, the
INTERPRETATION:
threshold can be set to Auto to show the threshold
▪ POSITIVE CONTROL: a value of 26 for ORF1ab, 25 for N
line where you can manually input or adjust a
Gene, and 28 for RP. All three are interpreted as
threshold
Positive (+).
rRT-PCR Results
▪ NEGATIVE CONTROL: usually blank or dash lang.
Consider this as negative as there is no data/CT value.
▪ REMARKS: VALID
Interpretation of Results in Unknown RNAs
▪ There is a linear format and a log format
▪ The diagram above is an example of a log format
▪ (-) means CT value > 40 or undetermined
o Depends on the institution. Sometimes, If there is
a value for a specific gene (ex: 40.56) it is not
instantly released as negative.
o Run or Extraction is repeated in order to confirm
the result
o A good Negative result should have NO CT
VALUE
o Confirm low titer, low viral load, contamination
▪ (+) means CT value is less than or equal to 40
o The shape of the graph must be curved.
o There are instances that the CT value is less than
40 (ex: 15) but the graph is not as curved or
sigmoidal.
o Do not instantly release as positive. Confirm the
graph first.
o A good Positive result has a CT value of less than
40 with a sigmoidal curve graph
o May be due to the deterioration of reagent or
primers, or due to contamination
▪ In Sample 1 in the table, CY5, FAM, and ROX are all
positive. Hence, it is interpreted as POSITIVE
(SARSCoV-2 specific RNA detected)
▪ Sample 2 is positive for CY5 and ROX while negative
for FAM. This is also interpreted as POSITIVE.
o This is for Sansure Biotech IFU. Institutions would
want to repeat testing to make sure that it was
only able to amplify 1 gene because a really
strong positive would have both genes.
▪ Sample 3 is positive for CY5 and FAM while negative
for ROX = interpreted as POSITIVE
o Do not instantly report as POSITIVE. Confirm the
result first. After 2nd testing, if it renders the same
result, result can be released.
▪ Sample 4 is only positive for CY5 (IC). This is interpreted
as NEGATIVE; SARS-CoV-2 specific RNA not detected.
▪ Sample 5: all are negative
o Concentration too low, repeat required
o Possibly due to improper sample collection
o If rendered negative for the second time,
release it as INVALID (because IC is negative)
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 Interpretation of Results in Unknown RNA • NTC (orange box)– no amplification; N/A • U1 – 35 is considered (+)
• Positive controls (green box) – has amplification; • U2 – 29 is considered (+)
must have a CT value of less than or equal to 35 • U3 – no values considered (-)
o PC1 Interpretation
o PC2 • U1 – Positive
“The results in the graph for positive colors have good • U2 – Negative
values thus, a very sexy sigmoidal curve.” • U3 – Negative. Repeat since no
amplification. Regardless of positive or
Conclusion Amplification Results negative, internal control must have a value.
SARS-Cov-2- There is a typical S-shape
Positive amplification curve detected at
Limit of Detection
FAM and/or ROX channel, and the
amplification curve which is Sansure Biotech Protocol
detected at CY5 channel, Ct <40 • Limit of Detection: 200 copies per mL
SARS-Cov-2- There is no typical S-shape • Coefficient of Variation: < 5%
Negative amplification curve or Ct < 40 • Specific for 2019-Novel-Coronavirus
detected at FAM and/or ROX • No cross-contamination with other
channel, and the amplification coronaviruses (e.g. SARS-Cov, MERS, Influenza A
curve which is detected at CY5 and B, Influenza H1N1, H3N2, and other
channel, Ct <40 influenzas)
REMEMBER
The interpretation of results shows the graph: • Cy5 channel - Ct <40 in both positive and
• Orange – graph for ROX or N gene target gene negative result
• Purple – graph for CY5 • Always correlate the amplification curve and
• Blue – graph for FAM or ORF 1ab the cycle threshold values
The graph should be showing a “sexy curve” or sigmoidal
curve Let’s try to interpret:
• Noise – encircled at the lower left side
• Threshold lines – (black circle) every target gene
have different threshold levels, and it should be
set above the noise
• True amplification – sigmoidal curve
“If we put the threshold line below the noise, it will be able Graph: shows the inverse proportionality between the
to read the noise and we don’t want that kay it’s not true cycle threshold values and the amount of viral RNA in
amplification. You base your true amplification to the the sample
sigmoidal curve or the S curve shape of the graph and to • The lower the Ct value, the larger number of
the CT values as well.” viral RNA in the sample. (Only an estimate but
not the actual amount of viral RNA)
Day 1 (red): <10 is considered as strong positive
Day 14 (blue):
Day 22 (green)
Day 29 (yellow)
GOOD TO KNOW
If positive Ct values or if it falls within the ranges <30 or
ORF 1ab - FAM channel <25
• U1 – 30 is considered (+) • Positive samples will be considered for
• U2 and U3 – no values considered (-) genome sequencing. In there, it will be known
N gene if the positive samples are Delta or Omicron.
• U1 – 20 is considered (+) That is why we aliquot samples to send the
• U2 and U3 – no values considered (-) aliquoted samples to research institutions.
The table beside the graph above shows the result of: Internal Control (IC)

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 control replace test tubes
Troubleshooting reaction in use.
(1) Operator-related (OR) PCR tube not Ensure plates are
• Pipetting skills properly sealed sealed correctly.
• Contamination process
• Good laboratory practices
(2) Reagent/Kit-related (RR)
• Degradation of reagents
Common • Expiry of reagents
Sources of • Contamination of reagents
Error • Exposure to degrading
agents
(3) Machine/Software-related (SR)
• Probes or heating block of
PCR machine are destroyed. A successful real-time PCR experiment will have the Table: Even though only one target gene is positive, it
If heating block is not able to following characteristics: is still considered invalid negative control.
heat, call the manufacturer • Curves are all S-shaped • A good negative control must not have an
(1) Amplification in the No Template • Plateau height doesn’t matter amplification in both genes.
Control o Plateau height – resting phase or
• Due to contamination stage in PCR, where components B. Reagent-related
(2) No Amplification in the Positive are consumed Probable cause Corrective measures
Control • Curves are smooth Contamination of Use fresh tubes (snap-cap tube,
• Due to buffer mix preparation • Dilution series has expected spacing PCR Reagents PCR tubes have are sterile and
• Forgotten to add PC • Replicates are tightly clustered have been autoclaved) of
• Too little amount of required • Melt curve has one peak per product reagent and make a new
volume was added • aselines are relatively flat mastermix preparation; In making
(3) No Amplification in the Internal a new mastermix prep: good
Control I. Amplification in the NTC/NC laboratory practices (e.g. wearing
• Improper collection of gloves)
Common A. Operator-related:
Immediately cap reagent tubes if
Errors (4) No Amplification in all wells/tubes Probable Corrective measures
not in use; DO NOT LEAVE THE
Encountered • Due to buffer or mastermix cause
REAGENTS OPEN esp if it’s not in
preparation Contamination Practice careful techniques and
the laminar flow cabinet
(5) High noise at the start of acquired due to carry preparation of NTC tube/well (e.g.
Practice Proper pipetting
data over from immediately cap the NTC well prior
techniques (do not touch the
(6) Expected C1 is lower than expected neighboring to adding sample templates and
pipette tip! If it’s been in contact
(7) C1 increases for re-analyzed wells PC)
with any surface, replace with
templates over time Leave at least one well empty after
new tip. Even the smallest error
(8) Unusual amplification curves the NTC
can greatly affect the PCR results)
(9) Plateau of amplification curve is Contamination Review and implement proper
lower than expected from the laboratory practices including
Individually packed kit Pre-mixed kit
(10) Data appearing in selected wells environment disinfection of all work surfaces and
components: components:
Drifting baseline equipment
Swapping with fresh Entire kit must be
Use new tubes and nuclease-free
reagents possible (e.g replaced
filtered tips and tubes
Sansure Biotech Kit)
Troubleshooting
If the Contamination of Clean surfaces
fluorescent the extraction/ and instruments
signal is preparation area with aqueous
detected in detergents, wash
a negative lab coats, and

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 If you receive an unsealed kit, you can report to/notify the PCR protocol
your supervisor set-up
II. No Amplification in the PC Major PCR Ensure the use of nuclease-free
A. Operator-related Inhibition consumables
Probable cause Corrective measures Troubleshooting
No PC template Practice careful pipetting
was added to techniques (may be due to minute
the designated amount of the template—not
well enough volume)
Double-check that the PC
template is added to the
appropriate well; done after you
have dispensed your RNA
template (add equal volumes)
B. Reagent related Editing the baseline setting (Auto-calculated or user-
• Negative and Positive controls are all negative (-) defined?)
Probable cause Corrective measures
for Noises: Might be due to bubbles, dust, or dirt
PCR Inhibition Ensure the use of nuclease-free
• Invalid run—repeat extraction
consumables
Troubleshooting
Repeat the PCR run following strict
molecular biology techniques V. High Noise at the start of acquired data If the Components Use a new batch
Check the expiry of the reagents A. Operator-related fluorescent degraded
Probable cause Corrective measures signal does Poor quality of Repeat the test
Template Dilute template with nuclease- not display RNA samples with the neat
III. No Amplification for the IC
concentration free water and repeat PCR run the sigmoidal carrying extracted RNA
A. Operator-related
too high characteristic interferences (preferably
Probable cause Corrective measures manual extraction
Insufficient RNA Repeat RNA Extraction B. Software-related
method and 1:10
was extracted If no lC amplification still occurs Probable cause Corrective measures
dilution of the
prior to RT-PCR after repeated extraction, Improper Review each curve and their
extracted RNA
problem could be in the pre- baseline respective baseline settings on the
PCR Equipment Repeat the test or
analytical phase (sample adjustment on PCR software
failure contact
collection or transport) and re- PCR software Edit baseline settings if necessary
equipment
swabbing should be considered (e.g. too many
supplier
Review product insert regarding cycles included)
IMPORTANT
the type of IC used. Read the • The end user is required to review fluorescent curves
information for use before final interpretation.
o Ct 9 (very low) and at the fluorescent curve,
IV. No Amplification in all wells
it doesn’t have a sigmoidal characteristic. It
A. Operator-related
is not true amplification.
Probable cause Corrective measures • All positive curves should be typical S-shape
Missing Make sure that all PCR amplification curves. can be without plateau for
components in components are systematically
weak positive samples (Ct 38-40).
the PCR added in the preparation of the
o Strong positives at 20 cycles will have a
mastermix mastermix (forgot adding the PCR Abnormal Noise at the start of the
mix/ Enzyme mix) graph=Dancing graph cycle; sigmoidal characteristic
Wrong channels Ensure appropriate channels are True amplification must be sigmoidal (S-shaped)
selected during selected in the PCR plate set-up
PCR plate set-up prior to run
Plate read/Data Ensure that the plate read/data
collection step collection step is included in the
was not added in protocol set-up

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 Troubleshooting • Buckingham. L. 20l9. Molecular Diagnostics: • The reagents must be
• If curves are not Fundamentals. Methods and Clinical Applications. Third placed on a cold rack to
smooth, suspect: Edition. FA. Davis Company, Illinois. USA maintain the quality of the
- Poor pipetting • Corman VM. Landt O, Kaiser M. et al. Detection of 2019 reagents.
(bubbles) novel coronavirus (2019-nCoV) by real-time RT-PCR.
- Sample Euro Surveill 2020;25. • We also prepare a small snap
evaporation • Lodish, HF et al. 20 I 6. Molecular Cell Biology. Eighth cap tube for the Master Mix.
- Poor assay (low edition.VV.H. Freeman-Macmillan Learning. New York, This is where you will mix your
fluorescence USA enzyme and PCR mix
reagents)
- System Real Time PCR: Laboratory Video 4. Allow complete thawing of buffers and reagents
malfunction 1. Get your reagent inside the 10 (enzyme & PCR mix)
(line noise) to 30C refrigerator along with • Proceed to pulse vortex and spin down
cold racks. • Reminder: Do not pulse vortex your enzyme mix
• If baselines are not flat, suspect: but only spin down immediately.
- Sample evaporation PCR Mix Enzyme Mix
- Bubbles Buffer preparation
• Pulse vortex • Spin down only
- Reagents not thoroughly mixed 2. In the buffer/reagent/mastermix preparation room:
- Make sure that
- Baseline “window” not properly set • Before entering the room, make sure to wear the
the PCR mix is
proper PPE (disposable gowns, gloves, bonnet,
homogenous
and facemask).
• Prepare your assay worksheet
− For the activity, there
are 5 unknown
samples and we’re
going to use 3
controls
• Common themes in troubleshooting − Incorporate the NTC, which is included in • Spin down
- Care in pipetting your 3 controls. So, we will have one negative
- Care in choice of plastics and sealing the control, one NTC (no template control), and
plates one positive control.
- Care in experimental design • This is how your plate looks
- Use of positive and negative controls like. Since we are going to
use 8 total reactions (5
Troubleshooting: Considerations unknowns & 3 controls),
let’s add 2 extra reactions 5. Continue the procedure inside the Laminar Flow PCR
Results are for the detection of 2019-nCoV RNA:
because we need a cabinet.
• Positive results are indicative of active infection with
2019-nCoV but do not rule out bacterial infection or minimum of 10 reactions
co-infection with other viruses. The agent detected for buffer preparation.
may not be the definite cause of disease. 3. So, we have here the Sansure novel
• Negative results do not preclude 2019-nCoV coronavirus nucleic acid diagnostic
infection and should not be used as the sole basis for kit from Sansure Biotech.
treatment or other patient management decisions. • We already removed the positive
Negative results must be combined with clinical & negative control and store it
observations, patient history, and epidemiological inside the refrigerator.
information. Enzyme Mix PCR Mix
• The cabinet lights are off because the enzymes,
References: probes, and reagents are light sensitive and to
• Philippine Genome Center make sure that they will not
• National Institute of Molecular Biology and degrade.
Biotechnology University of the Philippines • We placed the assay worksheet on
• Sansure Biotech lFU the cabinet so that we have a
reference guide to how much

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 enzyme & PCR mix we are going to add as well as 9. We have here your 11. Define Samples:
the number of samples. extracted RNA Sample Name
• For 5 unknowns + 3 samples and your NTC
controls + 2 extra controls. We will be NC
reactions, we need to dispensing 20 µL of U1
prepare 260 µL of PCR the samples except U2
mix and 40 µL of to your well A (NTC) U3
enzyme mix (good for Master U4
Well Extracted RNA
10 reactions) using a Mix U5
1000 µL micropipette A – NTC 30 µL --- PC
for the PCR mix and a 100 µL micropipette for the B – (-)
30 µL --- 12. Assign Targets and Samples:
enzyme mix. Add both to your master mix tube Control
then pipette mix (initial mixing). 20 µL – from unknown
• Pulse vortex your Master Mix to mix your enzymes C – U1 30 µL In your well A is your
sample 1
and PCR and then spin down with a balance (like NTC, which is your
20 µL – from unknown negative control, so we
in a centrifuge). D – U2 30 µL
sample 2 check this one
6. Dispense 30 µL of your Master Mix for your 20 µL RNA 20 µL – from unknown
E – U3 30 µL (negative control)
template. sample 3
• We can use a full-plate 20 µL – from unknown
PCR. F – U4 30 µL
sample 4 For your well B, which is
• Dispense 30 µL of Master 20 µL – from unknown the negative control,
Mix per well. G – U5 30 µL
sample 5 we also check this
7. This is how your PCR strip will H – (+) (negative control).
actually look like after you 30 µL ---
Control
dispense. Each well will have • After RNA template addition,
30 µL of Master Mix. you spin down, and just to
• The caps are not fully
make sure there are no For well C, D, E, F, and
closed since we will
bubbles you can lightly flick G, are your unknowns
proceed to template addition later on and for the bottom of the tubes.
easier opening of the strip. so we check this
• Check the caps after to (unknown).
Template Addition prevent evaporation then cover with foil.
8. In the template addition room, we will put the RNA- Amplification
templates in the Master Mix.
10. Define Targets:
• If there are any • As you can see
delays, store the For well H, which is your
here, your positive control, we are
Master Mix inside a
targets are your going to check this
refrigerator (-10 to - ORF1ab, N
20C). Cover with foil (standard)
gene, RP gene
to avoid exposure to and their reporter dyes which is FAM, ROX, and
sunlight. Then put it inside the PCR cabinet. • In the “assign targets in the selected wells”, there
CY5, respectively.
• In the activity, we have 5 unknowns, 1 negative • Make sure that your “Quencher” is none. are legends:
and 1 positive control • For the color, you can set it to whatever you like − U – unknown
− In the assay worksheet, the NTC (no template − S – Standard (positive control)
Target Name Reporter Quencher Color
control) is included in the counting for the − N – negative control
ORF1ab FAM None Any
Master Mix.
N gene ROX None Any
− Will we still add a 20 µL template on your
NTC? RP CY5 None Any
o No, you won’t add anything to the NTC
except your Master Mix. This is to make
sure that there is no contamination in
your buffer mix preparation and
template addition since it does not
contain any RNA or controls. It also
serves as a negative control.

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 18. Since your FAM is only the reporter dye for ORF1ab,
ROX for N gene, and Cy5 for RP, in order for us to know
if there is amplification for all genes, we have to click
all.

13. In the “Run

method” tab
under the plate
setup, check the
thermocycling
conditions.
• Upon clicking, now you can individually check if
each of the sample/unknowns have
14. The reaction setup is already inputted by the machine. amplification, thus called as “amplification plot”.
• As you can see here (red rectangle) is the • Your NTX should not have any amplification.
reaction volume per well which is 50 µL, 30 of • Your Cy5 is the reporter of your fluorophore for
which is your master mix and 20 for the RNA your RP, which is your human RNase P gene, which
template. indicates that your sample came from a human
being.
• In well D12,
there is an
amplification
on your FAM,
ROX, and Cy5,
so we can list
this one as
positive.
15. Now this is your − There are cut-off values for your Ct values
amplification which also depends on the manufacturer’s
plot, where the instruction.
graph will form a − But for Sansure, we have a cut-off of 40. So,
sigmoidal curve. for this well, we can consider this one as
positive since both genes (ORF1ab & N
16. After template addition, place your PCR strip inside gene) are present with Ct values of 37.34 &
your PCR machine. 34.46, respectively.
• In actual setting, the PCR strip is
placed in the middle to be
balanced.
• But for the activity, it is placed
in the first strip (first column).
17. Close the PCR machine
and start the run.

Interpretation of Results

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