Comparison of Real-Time PCR and Conventional PCR by

Identifying Genomic DNA of Bovine and Porcine

Ahlam Inayatullah1, Annisa Fatmawati2, Emelda2, Muhammad Abdurrahman Munir2*

1
Faculty of Science and Technology, Universiti Sains Islam Malaysia, Bandar Baru Nilai, Nilai, Negeri Sembi-
lan, 71800, Malaysia
2
Department of Pharmacy, Faculty of Health Science, Universitas Alma Ata, Bantul, Daerah Istimewa Yogya-
karta, 55183, Indonesia
*Corresponding Author: muhammad@almaata.ac.id

ARTICLE INFO Abstract

Article History: Bovine and porcine are poultry meat that is consumed worldwide,
Received date: 16 June 2021 particularly in Southeast Asia. Both of them are prone to food
Revised date: 25 August 2021 counterfeit owing to several factors such as price, appetite and Halal
Accepted date: 27 September 2021 status. Sensitive and selective analytical methods are required to
Available online at: November 2021 control meat products distributed to markets. This paper studied
the sensitivity between real-time and conventional PCR. Bovine
Keywords:
and porcine were used as the sample to verify the sensitivity of the
Bovine, Porcine, PCR, DNA.
method. The result of the study found that the assays did not show
a specific difference during DNA analysis of bovine and porcine. In
conventional PCR, two pairs of DNA primers targeted cytochrome
b (Cyt b) were analyzed, resulting in 120 and 131 amplicons,
respectively. While qPCR was applied to analyze porcine and bovine
DNA. The detection limit of qPCR after porcine and bovine analysis
was 0.004 and 0.007 µg/µL, respectively. Results demonstrated
that the qPCR was reliable for verifying porcine and bovine DNA
compared to conventional PCR. Furthermore, the study concluded
that the developed assay could easily identify porcine and bovine
tissue in food products in low resource areas.

© 2021 Jurnal Kimia Terapan Indonesia. This is an open access article

under the CC BY-NC-SA license (https://creativecommons.org/licenses/
by-nc-sa/4.0/).

1. INTRODUCTION of certain country related to Halal food, where

Nowadays, analysis of food products is impera- Islamic law stringently prohibits the consumption
tive to identify the quantity and quality of food, of specific meat products (e.g., porcine products)
preventing food adulteration and promoting food (Dolch et al. 2020; Zia et al. 2020; Kang et al.
safety. Meat species analysis becomes a persistent 2021). Those reasons should be considered to
issue that must be handled for several reasons satisfy and protect consumers. There are several
such as (a) the quantity of meat that is different approaches to detect meat species in foods, such
compared on the product label, (b) substitute as genomics (Wang et al. 2019; Sultana et al.
high-quality meats partially, even for some 2020), spectroscopy (Sankar et al. 2020; Cebi
cases convert entirely with low-quality then put et al. 2019), chromatography (Ekasary et al.
counterfeit label deliberately before distributed to 2018; Sha et al. 2018), morphology (Labrooy et
markets, (c) the concentration of meat inside non al. 2018; Zhang et al. 2019), immunochemical
– meat products and (d) to follow the regulation (Tukiran et al. 2016) and proteomics (Chis &
Vodnar 2019; Wang et al. 2019). The genomic
J.Kim.Terap.Indones. 23(2), p-ISSN: 0853-2788, e-ISSN:2527-7669
pp. 63–71, November 2021

method with the PCR technique has been used study. In comparison, 23 (17.7%) of the clinical
by Li et al. (2021) to detect the species content in specimens tested positive for GBS colonization
the product accurately. Furthermore, living organ- with conventional PCR, and 38 (29.2%) tested
isms have their own DNA’ molecules that very positive with qPCR. The study concluded that
unique for each organism (Williams et al. 2020; the qPCR technique had a better performance in
Zulch et al. 2020). PCR technique is strongly identifying positive SGB clinical specimens than
selective and sensitive by multiplying nucleotide conventional PCR.
sequence-specific nucleotides exponentially in The basic of PCR technique is the selection of
vitro (Toohey – Kurth et al. 2020). primer to be used. The specific primer will be at-
The concept of PCR requires the specific tached to the region-specific to the DNA template
DNA sequence parts to be multiplied before the and amplified into a new strand. A precise primer
multiplication process can be done. The sequence design is required to produce specific primers that
is imperative to provide a primer where the short match the target amplification. To detect porcine
oligonucleotide sequence initiates the DNA and bovine DNA, one of the genes that can be
synthesis in a polymerase chain reaction (Li et used as a specific marker is the Cytochrome b
al. 2020). Furthermore, the reaction is followed gene (cyt b). Cytochrome b (mt Cytb) gene has
by a heating machine that provides thermal been proved as an efficient tool with high power
conditions for amplification purposes (Mancini of discrimination for species identification and
et al. 2020). The process inside the PCR machine characterization in both taxonomy and forensic
is divided into three steps such as denaturation science (Saif et al., 2012). It is also used in stud-
(double-stranded DNA separation), annealing and ies of molecular evolution (Prusak et al., 2004).
extension (primer elongation) (Liu et al. 2020). The gene length is 1140 bp and has some stable
The PCR method generally applied sequences used to suggest universal primers for
nowadays is real–time PCR or known as quan- typical PCR-based methods (Parson et al., 2000).
titative polymerase chain reaction (qPCR). It Method validation is the practical process of
has advantages compared to conventional PCR determining the suitability of a method for pro-
(cPCR) that can continuously record the products viding analytical data that is fit for the intended
accumulation during the cycle where cPCR still purpose. For any method to produce meaningful
relies on agarose gel electrophoresis to determine and reliable data, some performances checks
the amplicons (Dorlass et al. 2020). The quantity should be made before the method is applied to
of qPCR is calculated by applying the threshold a real sample (Ali et al., 2012). There are many
cycle (Ct) based on fluorescent intensity induced performances characteristics that can potentially
by noise (background fluorescence). Noise causes be investigated for a particular method, some of
the attachment of DNA solution isolates along which are used in this study.
with PCR reagents in the tube wall (Karami et al. Specificity is the ability to measure only cer-
2020). Several advantages of qPCR have been tain substances carefully and thoroughly with the
studied and published by researchers (Yang et al. other components present in the sample matrix
2020; Cellier et al. 2020; Guo & Pooler 2020; (Brown, 2005). The component of the specificity
Ahmed et al. 2020; Kim et al. 2020; Farhan et al. test in this study is the oligonucleotide primer.
2020; Zheng et al. 2020) owing to the high preci- The nucleotide sequence is specified using NCBI
sion and accuracy during the detection in each Blast software to confirm the species origin of the
cycle (exponential phase) compared to cPCR that sequence of oligonucleotide primer.
determines in the final phase of amplification (the
The study determined the detection limit
plateau phase) due to the accuracy is lower than
(DL) of each PCR method under their optimal
qPCR (Karimi et al. 2020). Ferreira et al. (2018)
conditions using five total series of genomic
studied the assessment of conventional PCR and
DNA. Practically there are several ways of de-
real-time PCR for screening Streptococcus aga-
termining the detection limit of a method. The
lactiae in pregnant women, and the result shown
analyte is typically diluted serially in qualitative
among the 130 clinical specimens used in the

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analysis until it can no longer be detected reliably AAT CCT AAC AGG CCT G -3’ (forward) and 5’
using the method. The efficiency is additional -CGT TTG CAT GTA GAT AGC GAA TAA C-3’
information to show how accurate and reliable (reverse). The primers used for species-specific
real-time PCR compare to conventional PCR. amplification of bovine genomic DNA were 5’
The study compares both assays using a descrip- -CCC GAT TCT TCG CTT TCC AT-3’ (forward)
tive approach. The different techniques of both and 5’ -CTA CGT CTG AGG AAA TTC CTG
assays make it is impossible to compare quanti- TTG-3’ (reverse). Custom synthetic oligonucle-
tatively. This study aimed to introduce a suitable otide primers were obtained from IDT. The sizes
and sensitive technique between real-time and of the expected porcine and bovine amplicons
conventional PCR. were small (131 bp and 120 bp, respectively),
which were essential given the extent of DNA
2. EXPERIMENTAL SECTION degradation possible in highly processed prod-
ucts.
2.1 Materials and Method
2.1.1 Sample Preparation 2.1.3 Real-Time PCR Assay
Two different genomic DNAs animal species Genomic DNA of cattle and pig were diluted and
samples from porcine and bovine sources were subjected to the SYBR green-based PCR. The
obtained from Eurofins, respectively (Table 1). reaction was carried out using the SsoAdvanced
Porcine and bovine DNA were prepared by dis- universal SYBR Green supermix kit (BioRad,
solving the control DNAs of porcine and bovine USA) 10.4 µL, with the 10 µM and 0.4 µL of
in distilled water with series of concentrations reverse and forward primer, and a DNA concen-
10-1 - 10-5 ng/µl. tration adjusted to 2 µL. The supermix kit is a
reagent to help the PCR assay to send a signal
Table 1. General description of control DNAs em- and stabilize the PCR assay. The kit contains
ployed in this study dNTPs, MgCl2, SYBR green I dye, enhancers
Con-
and stabilizers. Amplification was performed in
Genomic Company the StepOnePlus System (Applied Biosystems,
Batch no. centra-
Type Name
tion USA) under the following conditions of tempera-
Genomic Eurofins 5222581306 150 mL ture and cycling: an initial cycle at 95 ◦C for 10
DNA of [ng/µl] minutes (pre denaturation stage); 40 cycles at 95
Cattle
◦C for 10s (denaturation stage), and continued at
(Bos
taurus) 63 ◦C for 45s (annealing and elongation stage).
Genomic Eurofins 5212581501 150 mL The stage was continued to measure melting
DNA of [1 ng/ temperature for 1 cycle at 95 ◦C in 15s and then
Pig (Sus µl] cooled at 60 ◦C for 30 s. This stage collects the
scrofa fluorescence signal at the end of each cycle. The
domes-
tica) results were analyzed using the cycle threshold
(Ct) and Tm.
2.1.2 Primer Design
A DNA sequence of the mitochondrial genome 2.1.4 Conventional PCR assay
was obtained from Tanabe et al. (2007). Regions This study used the same reaction as described
with high similarity were chosen for primer bind- by real-time PCR assay. Amplification was
ing sites in the area coding for the Cytochrome performed in the T100 Thermal Cycler (BioRad,
b gene. The theoretical specificity of all primers USA). The performance was begun at 95oC for
was checked with the Primer-BLAST software 7 minutes and continued with the denaturation
(Basic Local Alignment Search Tool, NCBI). The stage at a similar temperature for 30 seconds.
primers used for species-specific amplification The second stage was the annealing stage, where
of porcine genomic DNA were 5’ -CTT GCA primer was designed to anneal the single-stranded

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J.Kim.Terap.Indones. 23(2), p-ISSN: 0853-2788, e-ISSN:2527-7669
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DNA target. While for porcine primer, the an- Table 2. Mean Ct Values Obtained with the Real-
nealing stage was at 63oC whereas the annealing time PCR
stage for bovine primer was at 61oC. The stage Con- Porcine Primer Bovine Primer
was repeated for 40 cycles. Furthermore, the cen-
elongation was the third step that must be oc- tration Mean % Mean %
(ng/ ± SD RSD ± SD RSD
curred at temperature 72oC and proceeded to the μl)
last step at a similar temperature for 7 minutes. 10-1 18.14 ± 0.79 3.99 19.84 ± 1.58 8.69
Afterward, the PCR products were determined
10-2 21.92 ± 1.07 4.60 23.22 ± 2.13 9.70
using electrophoresis in 1% agarose gels in 1x
10 -3
24.77 ± 1.31 4.90 26.79 ± 1.14 4.60
TAE buffer followed by gel green staining and
visualization under UV light transillumination. 10 -4
28.11 ± 2.07 7.12 29.02 ± 0.37 1.31
The 1 kb DNA ladder marker was applied to 10 -5
31.94 ± 0.66 1.90 34.46 ± 0.75 2.36
determine the size of all DNA fragments. SD: standard deviation; RSD: relative standard deviation

2.1.5 Statistical Analysis The correlation between the Ct value and

The most effective means to measure assay the log concentration of five-fold dilution using
performance is by constructing a standard curve Pearson’s analysis showed a negative correlation
from a serial dilution template (Hofmann et al., (Porcine: -3.386 and Bovine: -3.569) with a p
1999). A type-I error (α) of 5% and equivalent value <0.05 (Figure 1), indicating the higher the
95% coverage for genomic DNA was used for concentration of DNA in the DNA sample, the
all analyses. Correlation between Ct-values lower the Ct value obtained. On the other hand,
against the log of the target concentration was the lower the DNA concentration, the higher the
calculated using Pearson’s correlation coefficient Ct value.
and expressed as its associated R2 (which is the
squared correlation, the percentage of variance
explained or in common). Efficiency can be
calculated according to the equation: 10(-1/slope) –
1. The calculation was performed in Microsoft
Excel 2016 (Redmond, USA).

3. RESULTS AND DISCUSSION

Figure 1. Standard Curves of 5-fold Dilutions of Por-
3.1 Real-time PCR cine and Bovine DNA
The diagnostic status of the sample was deter-
mined based on the obtained Ct value. The range The real-time quantitative PCR method
of Ct values for SYBR Green dye in porcine proposed in this study allowed us to detect each
samples was 18.14 – 31.94. While the bovine DNA over a wide range. The amplification of
sample was 19.84 – 34.46. The Ct values range each DNA species was clearly observed in a
was not exceeding more than 40, and it may be range between 0.1-0.00001 ng/µl. In the case of
stated that the Ct values obtained from the two 0.00001 of each DNA species, amplification was
primers are acceptable (Table 2). apparently detected. Hence, we concluded that
the limit of detection of those porcine DNA and
Five-fold dilution series of 10−1–10−5 gave stan-
bovine DNA species were10−5 ng/µl since it has
dard curves for detecting the genomic DNA of
shown an amplification curve for this concentra-
each bovine and porcine in real-time PCR. The
tion.
one-step real-time PCR system’s efficiency and
R2 values were 97.4% and 0.962 for porcine, and For the primer specificity test for bovine and
90.6% and 0.995 for bovine, respectively (Figure porcine, both primers showed specific results
1). which can be seen in Figure 2 for the porcine

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J.Kim.Terap.Indones. 23(2), p-ISSN: 0853-2788, e-ISSN:2527-7669
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(a) (b)
Figure 2. The amplification plot of Porcine DNA using qPCR (a) amplification plot and (b) Melting Curve

(a) (b)
Figure 3. The amplification plot of Porcine DNA using qPCR (a) amplification plot and (b) Melting Curve

test and Figure 3 for the bovine test where the peared, which was known to come from a nega-
negative control sample did not experience an tive control sample (bovine DNA) at 79oC that
increase in the amplification curve. This shows indicated that non-specific amplification products
that the SYBR Green method with bovine and had been formed. The event is often referred to as
pig primers can amplify their respective DNA mispriming or primer-dimer. Primer-dimer is the
identities specifically. The specificity of the formation of a secondary structure caused by the
amplification process using SYBR Green can be annealing of similar primers or dissimilar prim-
analyzed through Melting peaks. A specific am- ers, such as between forward primers and reverse
plification process will produce one type of peak primer complements. Meanwhile, mispriming is
with the same Tm value (Figure 2 and Figure 3). the attachment of primers outside the target DNA
However, in porcine DNA testing, a Tm peak ap- sequence (Ponchel, 2007).

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(a)

(b)
Figure 4. Visualization of conventional PCR of (a) bovine and (b) porcine. Gel analysis of the Conventional PCR
products of 5-fold dilutions of bovine and porcine DNA to determine sensitivity and specificity. Lane M, Ampli-
Size 300-10,000 base pairs (bp) in 1 Kb increments. Lane 1-2: 0.1 ng/μL: Lane 3-4: 0.01 ng/ μL; Lane 5-6: 0.001
ng/ μL; Lane 7-8: 0.0001 ng/ μL; Lanes 9-10: 0.00001 ng/ μL; Lane 11-13: negative control; Lanes 14: blank.

3.2 Conventional PCR 4b. From Figure 4a it can be seen that specific
primers for bovine can only amplify DNA se-
The results of DNA amplification with conven-
quences in bovine species and cannot amplify
tional PCR were described in gel documentation
DNA sequences in pig species (Lane 11, 12 and
(Figure 4) and showed that the genomic bands
13). Different results were obtained for porcine
in bovine and porcine DNA were clearly visible
primers where in Figure 4b amplification occurs
without smear. It can be concluded the DNA of
on lanes 11 and 12 (Bovine DNA). Improper
cows and pigs has a high purity in low concentra-
annealing temperature can cause DNA not to be
tions. Amplification was carried out simultane-
amplified or miss-priming during amplification.
ously based on the specifications of the DNA
Thus, re-specification of pig primers was tested
being tested. The gel documentation was carried
at annealing temperatures of 60, 61 and 62oC
out twice for each DNA specification due to the
and reduced the number of PCR cycles to 30.
insufficient number of holes in the gel comb in
However, the same result is seen on gel. The
one process.
porcine primer can not specifically identify the
The specificity test of the primers using porcine DNA.
conventional PCR was shown in Figures 4a and

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Although in sensitivity test porcine primers Chis, L. M., Vodnar, D. C. (2019). Detection of the
can detect porcine DNA in the concentration of species of origin for pork, chicken and beef in
10-5 ng/20 µl (Figure 4b), while bovine primers meat food products by real-time PCR. Safety, 5,
83. doi.org/ 10.3390/safety5040083.
are only sensitive to the presence of bovine DNA
Dolch, K., Andree, S., Schwagele, F. (2020). Compari-
to concentration of 10-4 ng / 20 µl (Figure 4a),
osn of real – time PCR quantification methods
The porcine primer designed by Tanabe (2007) in the identification of poultry species in meat
was not effective and efficient to identify porcine products. Foods, 9 (8), 1049. doi.org/10.3390/
DNA due to the lack of specific porcine detection. foods9081049
Dorlass, E.G., Monteiro, C.O., Viana, A.O., Soares,
4. CONCLUSION C. P., Machado, R. R. G., Oliveira, D. B. L. et
al. (2020). Lower cost alternatives for molecular
According to the application of qPCR and cPCR, diagnosis of Covid – 19: conventional RT – PCR
both showed satisfactory sensitivity during the and SYBR green – based RT – qPCR. Brazilian
analysis of porcine and bovine genomic DNA. Journal of Microbiology, 51, 1117–1123. doi.
Both assays can amplify to a sufficiently low org/10.1007/s42770-020-00347-5
concentration (10-4 - 10-5 ng/20 µl). Unfortu- Ekasary, A., Harmita, Maggadani, B. P. (2018). Opti-
nately, the specific test of porcine primers in mized high-performance liquid chromatography-
fluorescence detection method for the measure-
conventional PCR still requires optimization of
ment of glycine, proline, and hydroxyproline
annealing temperature. Optimization of annealing concentrations in porcine gelatin. International
temperature is one of the important parameter Journal of Applied Pharmaceutics, 10, 325–360.
criteria for the success of PCR. While each PCR doi.org/10.22159/ijap.2018.v10s1.72
method had its pros and cons, a final choice of Ferreira MB, de-Paris F, Paiva RM, Nunes LS. (2018).
the PCR method depends on the purpose of its Assessment of conventional PCR and real-time
application and the expected concentration of PCR compared to the gold standard method for
screening Streptococcus agalactiae in pregnant
species product.
women. Brazilian Journal Infection Disease,
22(6), 449-454. doi: 10.1016/j.bjid.2018.09.005
5. CONFLICTS OF INTEREST Hofmann, K., K Fischer, E Mueller and W Babel.
The authors declare that they have no known (1999). ELISA Test for Cooked Meat Species
Identification on Gelatin and Gelatin Products.
competing financial interests or personal relation-
Food/Nahrung Journal, 43, 406-409.
ships that could have appeared to influence the
Kang, S. J., Jang, C. S., Son, J. M., Hong, K. W.
work reported in this paper.
(2021). Comparison of seven commercial Taq-
Man master mixes and two real – time PCR plat-
6. ACKNOWLEDGEMENT forms regarding the rapid detection of porcine
DNA. Food Science of Animal Resources, 41 (1):
The authors wish to thank the Faculty of Science
85 – 94. doi.org/10.5851/kosfa.2020.e80
Technology, Universiti Sains Islam Malaysia
Karami, A., Hasani, M., Jalilian, F. A., Ezati, R. (2020).
(USIM) for providing the facilities to finish this Conventional PCR assisted single – component
study. assembly of spherical nucleic acids for simple
colorimetric detection of SARS – CoV – 2. Sen-
sors and Actuators B: Chemical, 328, 128971.
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