Article

A Practical Approach for Quantitative Polymerase Chain

Reaction, the Gold Standard in Microbiological Diagnosis
Tímea Mosolygó 1,2,† , Krisztián Laczi 3,† , Gabriella Spengler 1,2, * and Katalin Burián 1,2

1 Department of Medical Microbiology, Albert Szent-Györgyi Health Center and Albert Szent-Györgyi Medical
School, University of Szeged, Semmelweis utca 6, H-6725 Szeged, Hungary;
[email protected] (T.M.); [email protected] (K.B.)
2 MTA-SZTE Microbiology and Health Education Research Group, H-6720 Szeged, Hungary
3 Department of Biotechnology, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary;
[email protected]
* Correspondence: [email protected]; Tel.: +36-62-545-115
† These authors contributed equally to this work.

Abstract: From gene expression studies to identifying microbes, quantitative polymerase chain
reaction (qPCR) is widely used in research and medical diagnostics. In transmittable diseases like the
Ebola outbreak in West Africa (2014–2016), or the present SARS-CoV2 pandemic qPCR plays a key
role in the detection of infected patients. Although the technique itself is decades old with reliable
approaches (e.g., TaqMan assay) in the diagnosis of pathogens many people showed distrust in it
during the SARS-CoV2 outbreak. This came mainly from not understanding or misunderstanding the
principles of qPCR. This situation motivated us to design a simple laboratory practical class, in which
students have opportunities to understand the underlying principles of qPCR and its advantages
in microbiological diagnosis. Moreover, during the exercise, students can develop skills such as
handling experimental assays, and the ability to solve problems, discuss their observations. Finally,





 this activity brings them closer to the clinical practice and they can see the impact of the science on
Citation: Mosolygó, T.; Laczi, K.; real life. The class is addressed to undergraduate students of biological sciences.
Spengler, G.; Burián, K. A Practical
Approach for Quantitative Keywords: laboratory practical class; undergraduate teaching; qPCR
Polymerase Chain Reaction, the Gold
Standard in Microbiological
Diagnosis. Sci 2022, 4, 4. https://
doi.org/10.3390/sci4010004 1. Introduction
Academic Editor: Marcello Iriti The Polymerase Chain Reaction (PCR) was invented by Kary Mullis in 1983 and was
first used by the team of Cetus Corporation [1]. Since then, PCR technology has undergone
Received: 20 September 2021
a huge development, and it has become one of the most valuable and reliable methods used
Accepted: 7 January 2022
in diagnostics and bioscience. From the original end-point PCR, two distinct technologies
Published: 17 January 2022
have emerged for the quantification of nucleic acid concentration. The quantitative PCR
Publisher’s Note: MDPI stays neutral (qPCR), which is also known as real-time PCR, and the digital PCR (dPCR). All three
with regard to jurisdictional claims in technologies are based on the amplification of DNA with thermostable DNA dependent
published maps and institutional affil- DNA polymerase under 20–40 heat cycles. Each cycle starts with the denaturing of the
iations. DNA followed by the annealing of the oligonucleotide primers and finally the elongation
of the new strand. The theoretical product number at the end is the initial number of DNA
molecules ×2n where n is the number of cycles. The main difference between the three
methods is the way the product is detected. In a traditional end-point PCR, the product
Copyright: © 2022 by the authors.
can be detected with gel electrophoresis, and the amount of DNA is determined semi-
Licensee MDPI, Basel, Switzerland.
This article is an open access article
quantitatively based on the intensity of fluorescence in the gel; therefore, it is not suitable
distributed under the terms and
for quantification. In contrast, the qPCR can follow the concentration changes in real-time
conditions of the Creative Commons
by registering the level of fluorescence after every cycle. This allows the quantitation of
Attribution (CC BY) license (https:// genes, transcripts (cDNA), and microbes (by calculating genome equivalents dividing
creativecommons.org/licenses/by/ the measured copy number with the gene’s copy number in the genome) as well. The
4.0/). dPCR, on the other hand, kept the end-point detection, but it breaks down the reaction

Sci 2022, 4, 4. https://doi.org/10.3390/sci4010004 https://www.mdpi.com/journal/sci

Sci 2022, 4, 4 2 of 10

into hundreds or even thousands of micro reactions on microwell chips, or into droplets.
Every well/droplet contains exactly one or zero DNA molecules. After amplification,
the positive wells/droplets are counted based on a fluorescent signal; thus, the original
copy number of the sample can be determined without a calibration curve [2]. This is in
contrast with qPCR, where a calibration curve or an inner standard is used for quantitation.
Although dPCR has been found superior in precision and efficiency compared to qPCR,
and even the price/sample is lower for certain platforms, dPCR is more time consuming
and labor-intensive [3]. Therefore, qPCR is still preferred in clinical diagnostics, and has
become the gold standard of microbiological detection and quantification [4].
The qPCR operates with fluorescent dyes. As the product number increases cycle by
cycle, the emitted fluorescent light becomes stronger. There are two different types of qPCR,
the intercalating dye-based and probe-based. In case of a dye-based qPCR, the reason for
the increasing fluorescent intensity is based on the fluorescent dye’s ability to emit light
strongly only when it is intercalated into a double-stranded DNA (Figure 1a). As the cycle
number increases, more and more double-stranded DNA will be present in the sample so
more light will be emitted. In a probe-based qPCR, besides the primers, a fluorescently
labelled oligonucleotide probe is also added to the reaction. The probe hybridizes to the
template between the two primers on one strand. It is bound to the fluorophore and
the quencher, which quenches the fluorophore when they are in close vicinity. During
the synthesis of the new strand, the DNA polymerase digests the probe, setting free the
fluorophore and the quencher. The fluorophore moves away from the quencher and starts
emitting light (Figure 1b). In both cases, the increasing fluorescence can be described
with a sigmoid curve (Figure 1c). When the signal reaches the lower detection limit of the
instrument, the fluorescence starts to increase exponentially. During the exponential phase,
the reagents are exhausted and the curve goes into saturation. Where the curve crosses
the threshold line, the threshold cycle or quantification cycle (Ct or Cq ) is defined. The
threshold line is set by three rules: the threshold should be (1) above the background noise,
(2) on the log phase undisturbed by the plateau, and (3) at a point where all amplification
curves are parallel. The Ct value is proportional to the initial template concentration [5].
Probe-based qPCR is highly specific for the light is emitted only when the probe can
hybridize to the target sequence between the primers. Therefore, this method is considered
a gold standard in microbial diagnostics [6].
The intercalating fluorescent dyes such as SYBR® Green, SYTO dyes, EvaGreen® , etc.,
emit light when bound to the double-stranded DNA and illuminated with UV light [7].
Although this technique is cost-effective compared to the probe-based qPCR, the design
of the oligonucleotide primers should be carried out carefully. False products like primer
dimers may generate bias in quantification. Melting point analysis can indicate the artefact’s
presence in the samples, and the reaction or oligonucleotides can be optimized accordingly
(Figure 2). The melting peaks show the melting point of each of the products. With the
help of melting point analysis, false products can be caught without gel electrophoresis.
The melting point of the DNA depends basically on two main factors, the length and
the GC content of the fragment. Longer fragments have higher, shorter fragments have
lower, melting points, and fragments with the same length but higher GC content will
have higher melting points than their AT-rich counterparts. Therefore, melting point
analysis is more sensitive than gel electrophoresis for the detection of false products. A
computational method to correct qPCR results with the help of melting curves has recently
been proposed [8].
 is highly important in clinical diagnosis. In the case of SARS-CoV2 diagnosis, a significant
ratio of the samples is false-negative in the advanced stages of the disease due to inade-
quate sample collection [12].
The main purpose of this study was to design a simple laboratory practical approach
Sci 2022, 4, 4 to qPCR technology, in which students have opportunities to understand the underlying 3 of 10
principles of qPCR and its advantages in microbiological diagnosis.

1. Change
Figure 1. Changein influorescence
fluorescenceduring
duringqPCR:
qPCR: Panel (a)(a)
Panel depicts the the
depicts reason for increasing
reason fluorescence
for increasing fluores-
cence through
through the cycles
the cycles of qPCR of using
qPCRintercalating
using intercalating
dye. Thedye.
dye The
hasdye
onlyhas onlyfluorescence
weak weak fluorescence if not
if not bound
bound
by DNA.by At
DNA.
the At
endthe ofend
eachofcycle
each where
cycle where the products
the products are present
are present in double-stranded
in double-stranded form, form,
the
the intercalating dye binds the DNA and emits a strong light. More double-stranded
intercalating dye binds the DNA and emits a strong light. More double-stranded products mean products mean
stronger fluorescence.
stronger fluorescence. PanelPanel (b)
(b) shows
shows the
the principles
principles of of probe-based
probe-based qPCR.
qPCR. TheThe probe-based
probe-based qPCRqPCR
approach uses target-specific oligonucleotide probes labelled with a fluorescent dye and a quencher
approach uses target-specific oligonucleotide probes labelled with a fluorescent dye and a quencher
molecule. During the synthesis of the new strand, the DNA polymerase digests the probe, setting
molecule. During the synthesis of the new strand, the DNA polymerase digests the probe, setting
free the fluorophore and the quencher. The fluorophore moves away from the quencher and starts
free the fluorophore
emitting and the
light. Both qPCR quencher.
techniques The in
result fluorophore moves away
similar fluorescent from
curves, as the quencher
shown and(c).
on panel starts
The
emitting
curve canlight. Bothinto
be split qPCR techniques
3 main phases.result in similar
The first phasefluorescent curves,where
is the lag phase, as shown
onlyonthepanel (c). The
background
curve can
noise is be split In
detected. intothe3 main
second phases.
phase,The
the first phase is the
fluorescence lag phase,
increases where only
exponentially, theruns
and background
fast into
the plateau
noise phase.InIn
is detected. the the plateau
second phase,
phase, thethe fluorescence
fluorescence stops exponentially,
increases increasing dueand to the
runsdepleted
fast intorea-
the
gents. The
plateau threshold
phase. In the line cutsphase,
plateau the curve
the in the exponential
fluorescence stopsphase defining
increasing duethe threshold
to the depletedcycle.
reagents.
The threshold line cuts the curve in the exponential phase defining the threshold cycle.
 Sci 2022, 4, x FOR PEER REVIEW 4 of 11
Sci 2022, 4, 4 4 of 10

Figure
Figure 2.2.Correct
Correctand
andfalse
falsePCR
PCR products
products shown
shown on onaaschematic
schematicagarose
agarosegel (panel
gel (panel (a)) and
(a)) melting
and melting
peaks
peaks (panel(b)).
(panel (b)).In
Inwell
well 1,
1, aa DNA
DNA ladder
ladder is
isrun,
run,the
thesize
sizeofofthe
thebands is is
bands ononthethe
leftleft
side of the
side gel.gel.
of the
Well 2 contains the correct PCR product without any contamination. In wells 3–5, besides the correct
Well 2 contains the correct PCR product without any contamination. In wells 3–5, besides the correct
product, there are shorter and/or longer false products derived from mispriming or primer dimers.
product, therepeaks
The melting are shorter and/or longer
are derivatives falsecurves
of melting products derived
obtained frommelting
during mispriming or primer dimers.
point analysis.
The melting peaks are derivatives of melting curves obtained during melting point analysis.
2. Materials and Methods
The outbreak of SARS-CoV-2 sped up the development of new systems using qPCR,
2.1. Biological Material
e.g., in May of 2020, there were 81 kits and systems approved by the US FDA. Several
low-costEscherichia coli K-12
intercalating (ATCC 10798)
dye-based was cultivated
methods in Lysogeny
for SARS-CoV2 Broth were
diagnosis (10 g/L tryptone, to
published
5 g/L yeast extract, 10 g/L NaCl) at 37 °C shaken at 160 rpm until OD 600 = 1 (~8 × 108
overcome the financial struggle and elevate the throughput of virus diagnostics [9,10].
Incells/mL).
the case ofThe cells were collected
SARS-CoV-2 by centrifugation
PCR assays (10,000×
targeting ORF1a andg, ORF1b,
10 min) and
S and concentrated
N genes can
1.25 times in normal saline solution (9 g/L NaCl) resulting in a cell concentration
detect less than 10 genome equivalents [11]. It should be also mentioned that successful of ~109
cells/mL.is highly important in clinical diagnosis. In the case of SARS-CoV2 diagnosis, a
sampling
significant ratio of the samples is false-negative in the advanced stages of the disease due
to inadequate sample collection [12].
Sci 2022, 4, 4 5 of 10

The main purpose of this study was to design a simple laboratory practical approach
to qPCR technology, in which students have opportunities to understand the underlying
principles of qPCR and its advantages in microbiological diagnosis.

2. Materials and Methods

2.1. Biological Material
Escherichia coli K-12 (ATCC 10798) was cultivated in Lysogeny Broth (10 g/L tryptone,
5 g/L yeast extract, 10 g/L NaCl) at 37 ◦ C shaken at 160 rpm until OD600 = 1 (~8 × 108 cells/mL).
The cells were collected by centrifugation (10,000× g, 10 min) and concentrated 1.25 times
in normal saline solution (9 g/L NaCl) resulting in a cell concentration of ~109 cells/mL.

2.2. Isolation of DNA

DNA from 1 mL of the resuspended culture was extracted with QIAamp® DNA Mini
kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

2.3. Serial Dilution

An eight-step serial dilution with a scale of tens was performed on the purified
DNA. The most concentrated sample corresponds to 109 cells/mL while the most diluted
corresponds to 102 cells/mL.

2.4. Quantitative Polymerase Chain Reaction

The concentration of the E. coli in each dilution was analysed via qPCR. For the detec-
tion of the uidA gene, the following primers were used: 50 -CAACGAACTGAACTGGCAG-
30 and 50 -CATTACGCTGCGATGGAT-30 [13,14]. All primers were synthesized by Integrated
DNA Technologies Inc. (Montreal, Quebec, Canada). The primer hybridization sites on the
genomic DNA and the amplicon are shown on Figure 3. The qPCR was conducted with
the extracted and diluted DNA, primers (10 pmol/µL) and SYBR® Green JumpStartTM Taq
ReadyMix (Merck KGaA, Darmstadt, Germany) in a total volume of 20 µl, with a CFX96
Touch real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Thermal cycling was
initiated with a denaturation step of 10 min at 95 ◦ C, followed by 40 cycles each of 5 s
at 95 ◦ C, 20 s at 60 ◦ C and 25 s at 72 ◦ C. Cycle threshold (Ct ) values were determined by
automated threshold with Bio-Rad CFX Maestro Software version 2.2. Primer efficiency
was calculated automatically by the software based on the following equation:

Efficiency (%) = (10−1/slope − 1) × 100, (1)

where ‘slope’ is the slope of the calibration curve.

2.5. Exercise Design

The first step in this laboratory session was to divide the students into 4 groups, where
each group consisted of 3–4 students. Each group received 1 mL of E. coli suspension
(109 cells/mL), then the students carried out four exercises. First, they extracted DNA from
the original suspension of E. coli. In the second step, they serially diluted the purified
DNA, then they performed the qPCR. In the last step, the students involving the instructor
presented and discussed their results obtained from the qPCR graph.

2.6. Safety Considerations

At the beginning of the laboratory session, students were briefly informed about the
safety rules associated with working with biological samples, in order to avoid accidental
contamination. In addition, students were informed about laboratory waste disposal and
introduced to the location of the nearest fire extinguisher and first-aid kit. Disposable
gloves, safety goggles, and a laboratory coat must be worn in the laboratory.
Sci 2022, 4, 4Sci 2022, 4, x FOR PEER REVIEW 6 of 11 6 of 10

Figure Figure 3. Genomic

3. Genomic positioning
positioning of the
of the primer
primer bindingsites
binding sitesin
inthe
the E. coli
coliK12
K12substr.
substr.MG1655
MG1655 genome (NC_000913).
genome GreenGreen
(NC_000913). arrowsarrows
indicateindicate
the primer
thebinding
primersites,
binding sites,
purple purple
arrowsarrows indicate
indicate thestrands
the two two strands of the
of the PCRPCR product
product (genomicposition:
(genomic position:1,695,547–1,695,667
1,695,547–1,695,667 bp),
bp),yellow arrow
yellow show
arrow thethe
show genomic
genomicposition of uidA
position gene and
of uidA gene and the
the black lines are the two strands of the genomic DNA. The scale on the top indicates the genomic positions and should be read in base pairs.
black lines are the two strands of the genomic DNA. The scale on the top indicates the genomic positions and should be read in base pairs.
Sci 2022, 4, x FOR PEER REVIEW 7 of 11
Sci 2022, 4, 4 7 of 10

3. Results
The exercises
exercisesdesigned
designedwere wereassigned
assignedtoto
four
fourgroups
groups of students. TheThe
of students. workflow
workflow of the
of
laboratory procedure
the laboratory is summarized
procedure in Figure
is summarized 4. Students
in Figure in each
4. Students in group extracted
each group the DNA
extracted the
from
DNAthe fromoriginal E. coli E.
the original suspension, whichwhich
coli suspension, corresponds to 109to
corresponds bacterial cells. cells.
109 bacterial Then,Then,
they
serially diluted
they serially the purified
diluted DNA and
the purified DNA carried out the out
and carried qPCR theexperiments using each
qPCR experiments dilution.
using each
The groups
dilution. Theused the primers
groups used thetargeting the uidA gene
primers targeting (coding
the uidA genefor beta-glucuronidase)
(coding for beta-glucuron-and
amplify a 121-bps long part of the gene.
idase) and amplify a 121-bps long part of the gene.

Figure 4. Workflow of of the

the laboratory exercises. Cultures were grown on 37 ◦°C
laboratory exercises. C in an orbital shaker
incubator. Cells
Cellswere
werecollected
collectedbybycentrifugation,
centrifugation,then resuspended
then resuspended in saline solution.
in saline The The
solution. cell sus-
cell
pension waswas
suspension treated with
treated thethe
with QiaAmp
QiaAmpDNA
® ®
DNA Mini
Minikit’s
kit’slysis
lysissolution,
solution,then
thenthe
the cell
cell lysates were
lysates were
loaded to
loaded to the QIAamp®® Mini
the QIAamp Mini Spin
Spin Columns.
Columns. After
After washing
washing and and elution,
elution, serial
serial dilutions were made
dilutions were made
from the DNA samples by the factor of 10. The dilution series were utilized in the qPCR
from the DNA samples by the factor of 10. The dilution series were utilized in the qPCR reactions.
reactions.
Sci 2022, 4, 4 8 of 10

Sci 2022, 4, x FOR PEER REVIEW 8 of 11

Figure 5 shows the Ct values obtained by all groups and the calibration curve cal-
culatedFigure
from the results.
5 shows the CGroups 2 and 4 did
t values obtained by allnot haveand
groups amplification
the calibration in curve
the lowest
calcu- DNA
concentration sample. Group 1 had a
lated from the results. Groups 2 and 4 did not havehigher C t valueamplification in the lowest DNA con-and an
in the first and third reactions
abnormal
centration amplification
sample. Group curve.
1 hadThese
a higher four
Ct samples
value in the were firstdisclosed
and third from further
reactions and analysis
an
andabnormal
are not amplification
indicated in curve. FigureThese
2. The fourmean
samples were disclosed
Ct value of undilutedfrom further analysis (109 )
DNA samples
wasand are±not
7.08 indicated
0.52, while in theFigure
lowest2. The mean Ct value
concentrated samples (102 ) reached
of undiluted DNA samples (109) was with
the threshold
7.08 ± 0.52, while the lowest concentrated samples (10 2) reached the threshold with 31.07
31.07 ± 0.06 Ct . On average 3.39 cycles were between the neighboring dilutions in the same
± 0.06This
series. Ct. On
valueaverage 3.39 to
is close cycles
the were between3.32
theoretical the cycles
neighboring dilutions
difference in the same
between se-
the elements
ries. This value is close to the theoretical 3.32 cycles difference between the elements of a
of a 10 times dilution series. There was a slight difference between the samples of the
10 times dilution series. There was a slight difference between the samples of the groups
groups for the Ct value of the same dilution varies with an average of 0.87 cycles. This
for the Ct value of the same dilution varies with an average of 0.87 cycles. This deviation
deviation
betweenbetween the parallels
the parallels might by
might be caused be the
caused by the
pipetting pipetting
error of distincterror of distinct
students. students.
The cor-
Therelation
correlation between DNA concentration and cycle number was strong (R 2 = 0.993) and
between DNA concentration and cycle number was strong (R = 0.993) and all 2

all parallels
parallels fitfitthe
thetrendline
trendlinewell.well. Groups
Groups 3 and
3 and 4 measured
4 measured slightly
slightly lowerlower Ct values
Ct values for thefor the
samesameDNADNA concentration
concentrationcompared
compared to togroups
groups1 1andand 2 due
2 due to pipetting
to pipetting errors.errors.
All theAll
datathe data
werewereappropriate,
appropriate,building
building thethe calibration curvewith
calibration curve with thethe equation
equation = −3.420x
y = y−3.420x + 38.079
+ 38.079
(Figure
(Figure 5b);5b);
thetheprimer
primerefficiency
efficiency waswas96.06%.
96.06%.AtAt thethe
endend of the laboratory
of the laboratory exercise, the the
exercise,
students
students discussedtheir
discussed their results,
results, and
andinterpreted
interpreted thethe
calibration
calibration curve. To improve
curve. their their
To improve
understanding
understanding ofof
thethemain
mainpoint
pointof of the
the experiment,
experiment, students
studentshad had toto
estimate
estimate thethe
number
number of
of bacterial cells of unknown samples with the aid of the
bacterial cells of unknown samples with the aid of the equation of the line. equation of the line.

Figure 5. Results of the qPCR experiment: The sigmoid curves of the amplification are shown on
Figure 5. Results of the qPCR experiment: The sigmoid curves of the amplification are shown on
panel (a). RFU: relative fluorescence units. The horizontal red line represents the threshold. The
panel (a). RFU:
calibration relative
curve fluorescence
obtained units.series
from the dilution The horizontal
is shown on red line
panel (b).represents the threshold.
R2 = 0.993 where R2 is the The
calibration
square ofcurve obtainedcoefficient
the correlation from the dilution
and showsseries
how is shown
well on panel
our model R2 data
(b).real
fits the = 0.993 where
points.
2
R2 =R1 is the
2
square of the correlation coefficient and shows how well our model fits the real data points. R = 1 means
Sci 2022, 4, 4 9 of 10

a perfect fit, while lower values will represent less-perfect fits. Log starting quantity equals the
theoretical cell number of the samples. Melting curves of the samples are shown on panel (c). This
is a direct representation of fluorescence change in the samples in the function of the temperature.
As the temperature increases in the reaction tubes, the fluorescence drops gradually. The melting
temperature is found where the melting curve becomes precipitous. If the curve has more precipitous
sections, there will be multiple products with different melting temperatures in the sample. The
melting peaks on panel (d) show the melting point more spectacularly (see Figure 2 for description).

4. Discussion
Emerging and re-emerging infections are global public health concerns. Accurate
laboratory testing of the causative agent is essential for early discovery, isolation, and
treatment, in order to cut off the transmission route. The outbreak of SARS-CoV-2 has
drawn tremendous attention to the importance of clinical microbiology and the different
molecular and serological methods, such as rapid antigen tests, PCR, and evaluation of the
serum antibody levels. Viral RNA can be detected in the upper and lower respiratory tract,
stool, blood, and urine of SARS-CoV-2 infected patients. Due to its sensitivity and specificity
of qPCR is the preferred and most widely used method for detecting the presence of viral
nucleic acid in these samples [15]. Collective understanding of qPCR’s basic principles is
essential to increase trust in clinical diagnostics and pull out the venom of sceptic voices
who spread disinformation out of profit or gullibility.
Unfortunately, the introduction of different molecular methods to undergraduate
students in biology class is hampered by the lack of equipment and the cost of the reagents.
Moreover, the extremely rapid development of science has led to the fact that relatively few
biology teachers have practical experience of DNA techniques during their training.
This situation motivated us to design a simple laboratory practical class, in which
students have opportunities to understand the underlying principles of qPCR and its
advantages in microbiological diagnosis. Through this activity, students can perform DNA
extraction from E. coli and carry out qPCR amplification, which are routine diagnostical
tools in clinical microbiology. Moreover, during the exercise, students can develop skills
such as handling experimental assays, and the ability to solve problems and discuss their
observations. Finally, these exercises provide not only insight into the laboratory work, but
also connect theory to practice and stimulate interest and enjoyment of science.
At the end of the class, the student should be able to conclude that qPCR can be used
for the detection of nucleic acid in clinical samples, and the Ct value negatively correlates
with the number of the given microorganism. The designed experiment can be performed
over one laboratory class of 4 h or it can be divided into 3 sessions: (1) isolation of the DNA,
(2) dilution and PCR assay, and (3) interpretation of the results.
For its cost-effectiveness, we chose the fluorescent dye-based qPCR method to per-
form in this practical class. Although, in the diagnosis of SARS-CoV-2 infection, re-
verse transcription-coupled qPCR (RT-qPCR) is used, since this virus possesses an RNA
genome, in our designed protocol, DNA was used as templates, due to the instability of
RNA molecules.
The presented protocol was successfully implemented in a microbiological laboratory
course held for undergraduate students. The obtained results were appropriate building
the calibration curve, only four samples were disclosed, due to abnormal amplification.
This practical class can be extended to introduce additional molecular diagnostical
methods, such as isolation of RNA, multiplex qPCR, or RT-qPCR, where there is a reverse
transcription step before the qPCR.

5. Conclusions
The goal of this laboratory practical class was for high school students to learn how to
perform qPCR and to represent the results obtained in graphs. The laboratory exercises
presented in this study promotes active learning about the impact of molecular methods and
provides students with an opportunity to develop practical skills in the field of laboratory
Sci 2022, 4, 4 10 of 10

work. Finally, this activity brings the undergraduate students closer to the clinical practice
and they can see the importance of science.

Author Contributions: T.M. wrote parts related to the materials and methods, the discussion and
designed the experimental protocol. K.L. wrote parts related to the introduction and the results
and prepared the figures. G.S. came up with the original idea of the study and revised the final
manuscript. K.B. revised the final manuscript, supervised the study, and provided the funding. All
authors have read and agreed to the published version of the manuscript.
Funding: The research was funded by the Pedagogy Research Program of the Hungarian Academy
of Sciences (2016–2020).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors thank Veronika Péter-Kovács, Józsefné Kovács, Ildikó Vadászné Horváth,
and Brigitta Udvarhelyi (Tömörkény István High School, Szeged, Hungary) for pedagogical advice.
Conflicts of Interest: The authors declare no conflict of interest.

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