Applied Food Research 2 (2022) 100148

Contents lists available at ScienceDirect

Applied Food Research

journal homepage: www.elsevier.com/locate/afres

The development of real-time polymerase chain reaction for identification

of beef meatball
Abdul Rohman a,b,∗, Salmah Orbayinah c, Adam Hermawan b, Sismindari Sudjadi b,
Anjar Windarsih d, Sri Handayani d
a
Center of Excellence, Institute for Halal Industry & Systems, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
c
School of Pharmacy, Faculty of Medical and Health Sciences, Universitas Muhammadiyah Yogyakarta, Yogyakarta, Indonesia
d
Research Center for Food Technology and Processing (PRTPP), National Research and Innovation Agency (BRIN), Yogyakarta, 55861, Indonesia

a r t i c l e i n f o a b s t r a c t

Keywords: Real-time polymerase chain reaction (PCR), also known as quantitative PCR (qPCR), is a method of choice for the
Beef DNA confirmation of halal meats such as beef from non-halal meats such as pork through analysis of their deoxyribonu-
q PCR cleic acid (DNA). The objective of this study was to apply qPCR in combination with species-specific primer (SSP)
Meatballs
targeting in D-loop mitochondria genes for the identification of DNA extracted from beef and beef meatballs. DNA
Halal authentication
in raw meats and meatballs was isolated using Genomic DNA Mini Kit and the obtained DNAs were subjected to
SSP specificity, amplification efficiency (E), determination of limit of detection (LoD), and repeatability test. The
results showed that SSP designed was specific to beef DNA with LoD value of 100 ng beef DNA. The coefficient of
determination (R2 ) for the relationship between log DNA concentration and quantification cycle (Cq) along with
efficiency values were 0.884 and 97.1% respectively. The developed method is precise enough as indicated by the
low value of relative standard deviation (RSD) value of 0.77%. The qPCR method using SSP is also successfully
applied for analysis of beef meatballs commercially available. It can be concluded that qPCR using SSP targeting
on D-loop mitochondria could be proposed as a standard method for the confirmation beef meatballs.

1. Introduction
The issues on halal products are emerging in line with increased
awareness among the Muslim community worldwide (Adiarni & For-
tunella, 2018). Halal is an Arabic term meaning permissible and ac-
cording to Syariah (Islamic law). Concerning food and pharmaceutical
products, halal food can be understood as any food and pharmaceutical
products permissible to be consumed or to be used by Muslim societies
and free from any prohibited components such as pig derivatives. In-
donesia has stipulated Indonesian Act No. 33 year 2014 regarding Halal
Product Assurance in which food declared as halal must be halal certi-
fied, therefore, the certification of halal products is mandatory.
The halal assurance is the requirement for products to be consumed
by the Muslim community (Mursyidi, 2013). Non-halal components, es-
pecially pig derivates such as pork, lard, and porcine gelatins may be
found in food products labelled with halal products (Rohman & Pu-
tri, 2019), therefore, the availability of analytical methods detecting of
non-halal components for halal authentication analysis has been con-
tinuously developed (Mutalib et al., 2015). Meatballs are favourite food
consumed by the Indonesian community due to its pleasant taste. The
main components of meatballs are meats which are protein sources used
for human development. The price of beef, the meat used in meatballs,
is higher than other meats such as pork therefore unethical meatballs
producers may substitute beef with pork to reduce the production cost
(Rohman et al., 2016; Rohman et al., 2017). Some analytical methods
have been proposed and validated for analysis of meat types in meatballs
(Jamaludin et al., 2018) including enzyme-linked immunosorbent assay or
ELISA (Zia et al., 2020), liquid chromatography-tandem with mass spec-
trometer known as LC-MS (Sarah et al., 2016), gas chromatography us-
ing several detectors (Rohman & Fadzillah, 2018), Fourier transform in-
frared spectroscopy in combination with chemometrics (Pebriana et al.,
2017; Rohman & Che Man, 2010), and electronic nose (Peris & Escuder-
Gilabert, 2016; Tian et al., 2013). Some methods are lack in specificity
so that analytical methods based on DNA such as PCR are developed for
analysis of pig derivatives (Abbas et al., 2018).
Polymerase chain reaction (PCR) is a method of choice for iden-
tification of species with good specificity and accuracy because each
species has specific DNA (Arini et al., 2018). The development of PCR
into real-time PCR, also known as quantitative PCR, could quantify the
levels of DNA efficiently and effectively (López-Andreo et al., 2012).

∗
Corresponding author at: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia.
E-mail address: abdulkimfar@gmail.com (A. Rohman).

https://doi.org/10.1016/j.afres.2022.100148
Received 26 April 2022; Received in revised form 1 June 2022; Accepted 11 June 2022
Available online 13 June 2022
2772-5022/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
A. Rohman, S. Orbayinah, A. Hermawan et al.
Table 1
The meatballs containing the different levels of beef in a binary mixture with pork.
Concentration of beef (%) Beef (gram)
0 45
5 42.75
10 40.5
15 38.25
25 33.75
50 22.5
75 11.25
100 0 45
Compared to conventional PCR, real-time PCR (qPCR) have some ad-
vantages including its speed, reproducibility and its ability to perform
quantitative analysis of DNA targets. In addition, qPCR is more sensi-
tive and reproducible than conventional PCR, therefore, qPCR is widely
applied in diagnostic tasks including halal food authentication (Paiva-
Cavalcanti et al., 2010). Real-time PCR using species-specific primer
(SSP) (Doosti et al., 2014), TaqMan probe (Rojas et al., 2012), and du-
plex (Druml et al., 2015) has been used for analysis of DNA from pork.
Analysis of pork DNA using PCR may gain negative results which may
be coming from the unsuccessful extraction of DNA. Therefore, further
analysis of DNA from other meats is needed.
Some studies related to use of qPCR for quantitative analysis of
beef or beef components in food products have been carried out.
Chen et al. (2020) have used qPCR targeting mitochondrial cytb (cy-
tochrome b) fragment for identifcation and quantifcation of bovine in-
gredient in commercial meat products providing detection limit (DL) of
qPCR method of 0.025 ng DNA corresponding to relative DL of 0.002%
(w/w) of positive samples. Drummond et al. (2013) also employed qPCR
using species specific of bovine targeting on cytb for analysis and as-
sessed using the TaqMan probe and SYBR Green systems providing high
accuracy. However, in this study, DL was not reported. The combination
of qPCR and SYBR Green targeting on 12S rRNA gene has been devel-
oped for the quantitative detection of bovine tissues in food capable of
amplifing an 84-bp fragment corresponding to DNA target (Lee et al.,
2008). In this study, bovine DNA in the mixtures could be detected as
low as 0.1% of bovine tissues. In addition, using qPCR targeting on gene
myt ATPase 8–ATPase of bovine DNA using Taqman provide 0.001%
bovine materials (Lahiff et al., 2002). However, qPCR targeting D-loop
mitochondria genes is limited. Using different targeting genes, it is pos-
sible to compare the sensitivity of qPCR. This study aimed to develop
qPCR targeting on D-loop mitochondria genes for the identification and
quantification of DNA extracted from beef and beef meatballs.
2. Materials and methods
2.1. Materials
The meats including pork, beef, chicken, and lamb as well as
commercial meatballs were purchased from the local market in Yo-
gyakarta. The materials used for preparation of meatballs including
spices and flour were also bought from local markets. The laboratory-
prepared meatballs were performed according to Rahmania et al.
(Rahmania et al., 2015). To facilitate the construction of linear regres-
sion for determination of amplification efficiency, meatballs with differ-
ent levels of beef were prepared according to Table 1.
2.2. Primer designing
The primer sequences of D-loop mitochondrial for bovine (Bos
taurus) were designed using software from NCBI (http://www.ncbi.
nlm.nih.gov). The primer was synthesized by PT. Genetika (Jakarta, In-
donesia). The candidate primers obtained during this design was com-
piled in Table 2 assigned with primers of DA, DB, and DC.
Applied Food Research 2 (2022) 100148
Pork (gram) Flour and other components (gram)
0 5
2.25 5
4.5 5
6.75 5
11.25 5
22.5 5
33.75 5
5
2.3. DNA isolation
DNA in meat and meatballs was isolated using Mitochondria DNA
Isolation Kits from Biovision (USA). The procedure followed the in-
struction from the manufacturer’s instructions. The purity of isolated
DNA was analyzed using NanoQuant Spark Tecan (Switzerland) at wave-
lengths of 260 nm dan 280 nm. The calculation of DNA concentration
was based on absorbance value at 260 nm (A260 ) multiplied with ab-
sorbance constant of 50 μg/mL. The isolated DNA was considered pure
if the purity index obtained was in the range of 1.8-2.0. The DNA ob-
tained was used as template DNA during analysis using real-time PCR
(Kim & Kim, 2018).
[DNA concent rat ion](μg∕mL) = A260 x f p x 50μg∕mL
Purityindex =A260∕A280
2.4. Real-time polymerase chain reaction
The condition of RT-PCR used in this study consisted of the reaction
mixture (20 μL) comprising of 10 μL SYBR Green® universal PCR master
mix, 1 μL of reverse and forward primer (100 ng), 1 μL DNA template
(100 ng), and 7 μL of water-free nuclease. The temperature program
used was 94°C for 30 sec, which was followed by 30 cycles at 94°C for
5 sec for denaturation stage. The annealing temperature was optimized,
and the temperature for elongation stage was set at 72°C for 10 sec. The
melting curve was performed at temperature of 60°C–90°C with a slope
of 0.5°C/5 sec (Orbayinah et al., 2020).
2.5. Validation of real-time PCR
The used primer was subjected to evaluation by determining sev-
eral performance characteristics including specificity, amplification ef-
ficiency, and repeatability. Primer specificity was confirmed by ampli-
fying 100 ng/μL of extracted DNAs from pig, cow, chicken, and goat
along with No Template Control (NTC) or negative control. The effi-
ciency of DNA amplification was carried out by preparing laboratory-
prepared meatballs consisting of beef (halal meat) and pork (non-halal
meat) with concentration ratios of beef-pork of (100-0, 90-10, 75-25,
50-50, 25-75, and 0-100)%. The equation of linear regression obtained
by correlating the levels of DNA (x-axis) and Cycle threshold (Ct) value
(y-axis) was used for evaluating the efficiency value (E).
( )
E = 10(1∕slope) −1 x100
%E =(E − 1)x100%
The recommended value of E is 90–110% (Pratiwi et al., 2018). E-
value of <90 or ≥ 110% is unacceptable and indicates that further opti-
misation needs to be carried out (Rogers-Broadway & Karteris, 2015).
The determination of limit of detection (LoD) was based on ampli-
fication on 100% concentration of beef meatballs by serial dilution in
order to obtain a concentration of DNA at levels of 0.1, 1, 10, 100, 1,000,
2
A. Rohman, S. Orbayinah, A. Hermawan et al. Applied Food Research 2 (2022) 100148

Fig. 1. The amplification curve of DNA tem-

plate using primer MITB_3 at various annealing
temperatures of 49.2°C (A), 49.9°C (B), 50.9°C
(C), 52.1°C (D), 53.1°C (E), and 53.6°C (F).

Fig. 2. The specificity test of primer MITB_3

targetting D-Loop mitochondrial DNAs of beef
(A), chicken (B), lamb (C), pork (D), and nega-
tive control or no template control (E)

Table 2
The primer candidates targeting on D-loop mitochondrial of Bos taurus designed for identification of beef DNA.

Primers Sequence 5’ – 3’ Melting temperature (Tm) (o C) %GC Amplicon

MITB_1 F: 5′-TCT TCA GGG CCA TCT CAT CTA-3′ 62 47.6 115
R: 5′-CCA AAT GTA TGA CAG CAC AGT TAT G-3′ 62 40
MITB_2 134
F: 5′-ATC TCG ATG GAC TAA TGG CTA ATC-3′ 62 41.7
R: 5′-CAA TAG ATG CTC CGG GTC AG-3′ 62 55
MITB_3 114
F: 5′-GGA TGC TTG GAC TCA GCT ATG-3 62 52.4
R: 5′-TGA CTG TAA TGT CCA CGC TTA TC-3′ 62 43.5

Fig. 3. The amplification of beef‘s DNA at dif-

ferent levels of DNA, namely 0.1 pg (E), 1 ng
(D), 10 ng (C), 100 ng (B) and 1000 ng (A) us-
ing primer MITB_3 targeting D-Loop mitochon-
drial of Bos taurus.

3
A. Rohman, S. Orbayinah, A. Hermawan et al. Applied Food Research 2 (2022) 100148

Fig. 4. The calibration curve correlating the

different levels of log concentration of DNA
(x-axis) and Cq-values (y-axis) using primer
MITB_3 targetting on D-Loop mitochondrial of
Bos taurus.

and 10,000 pg. The repeatability of real-time PCR method was evaluated Table 3
by calculating relative standard deviation (RSD) of DNA amplification The levels and purity indexes of DNA isolated from meatballs samples.
(Ct value) from meatballs with 100% beef. Real-time PCR methods were Samples Concentration(ng/mL) Ratio A260 /A280
then used for analysis of commercial meatball samples commercially
Meatballs with pork 0% 1510 1.56
available in the market which are typically labelled as beef meatballs or
Meatballs with pork 5% 1350 1.39
halal meatballs (Orbayinah et al., 2019). Meatballs with pork 10% 1455 1.37
Meatballs with pork 15% 1065 1.39
3. Results and discussion Meatballs with pork 25% 815 1.55
Meatballs with pork 50% 2095 1.47
Meatballs with pork 75% 2280 1.58
Analysis of non-halal meat in meatballs such as pork using poly- Meatballs with pork 100% 2780 1.69
merase chain reaction (PCR) can gain negative results. There are two
possibilities from the negative result for pork DNA, namely (1) pork
DNA was not present or (2) the extraction of DNA was failed. As
a consequence, to anticipate the negative results due to unsuccess- of 49.2; 49.9; 50.9; 52.1; 53.1; and 53.6°C. Primer MITB_3 could am-
ful DNA extraction, the specific primer for beef DNA was designed. plify DNA at all temperatures as shown in Fig.1 The annealing tem-
Table 2 compiled the primer candidates for identification of beef in perature of 53.1°C was selected due to its capability to provide the
meatballs. In general, good primers had 18-24 base pairs with GC con- maximum fluorescence intensity with a minimum Cq value. The speci-
tents of 50-60%. Both primers (forward and reverse) are not comple- ficity test of primer was carried out by amplification of DNA isolate of
mented to each other to form dimer and the used primers should have raw meats of pork, chicken, beef, and lamb using an annealing tem-
a similar melting temperature. Among the designed primers in table 2, perature of 53.1°C. The amplification result exhibited that the fluores-
primer pairs of MITB_3 targetting on D-loop mitochondrial of Bos Tau- cence intensity of DNA extracted from beef, while there is no amplifica-
rus are ideal candidates because they meet the required primers. The tion observed for DNAs extracted from pork, chicken, and lamb (Fig.2).
designed primer was also subjected in silico using BLAST and the re- Therefore, it can be concluded that primer MITB_3 is specific for beef’s
sult showed that primer MITB_3 is a specific primer for DNA’s Bos DNA.
Taurus. The sensitivity of real-time PCR using primer MITB_3 was evaluated
The isolation of DNA was aimed to get a DNA template to be ampli- by determining the limit of detection (LoD) value of DNA extracted
fied using real-time PCR. DNA was isolated from raw meats and meats from meatball containing 100% of beef. Fig.3 revealed the amplifica-
in meatballs using Genomic DNA Mini Kit, PureLink. The extracted DNA tion curve of DNAs with different concentrations. At concentration 100
was qualitatively and quantitatively analyzed by determining the DNA’s ng, beef’s DNA is still amplified with Cq-value of 26.08. In addition, if
purity and DNA’s level based on the measurement of UV absorbance at DNA concentration was decreased at a level of <100 ng, the amplifi-
260 nm (A260 ) and 280 nm (A280 ), and the results were compiled in cation curve was not observed, therefore, the LoD value for analysis of
Table 3. The ratio (R-value) of A260 /A260 was in the range of 1.37-1.83 beef’s DNA using real-time PCR was 100 ng.
making DNA was suitable to be used as a DNA template. The ideal R- For evaluation of amplification efficiency of primer MITB_3, the beef
value was 1.8-2.0 in which R-value < 1.8 indicated DNA’s contamination meatballs with different levels of beef-mixture (0%, 1%, 5%, 10%, 20%,
with protein, while R-value >2.0 indicated that DNA was contaminated 50%, and 100%) were subjected to amplification and the calibration
with RNA.The discrepancy R-value from ideal value (1.8-2.0) should be curve which relate the DNA concentration and Cq value can be made.
use as basis for avoiding DNA over quantification (Lucena-Aguilar et al., There is a negative correlation between Cq-values and log concentration
2016). Codex Alimentarius Commission (2010) stated that high purity DNA (Fig.4). From this curve, the amplification efficiency (E) obtained
of the DNA allows optimum amplification thereby affecting the validity was 97.1% and this value was in the range of acceptable E-value (90-
of the PCR method. 105%) (Bio-Rad, 2006). The precision of real-time PCR for quantitative
The optimization of annealing temperature for DNA template to be analysis of beef’s DNA was evaluated using repeatability test and RSD-
amplified using primer MITB_3 was carried out by varying temperatures values of Cq. Using meatballs at 100% of beef concentration, Cq-values

4
A. Rohman, S. Orbayinah, A. Hermawan et al.
were in the range of 22.12-22.60 with the RSD of 0.77%. Low RSD values
indicated that the developed method was precise and applicable to be
used for analysis of commercial meatball samples. According to Codex
Alimentarius Commission (2010), RSD values must be lower to 25%.
Analysis of commercial meatball samples using designed primer MITB_3
indicated that all evaluated meatball samples use beef, as indicated that
DNAs were amplified.
4. Conclusion
Real-time PCR using species-specific primer targeting on D-loop mi-
tochondrial gene of Bos taurus has been successfully used for identifi-
cation and confirmation of beef DNA in beef meatball for halal authen-
ticity. The developed method is specific in which the designed primer
only amplifies beef DNA and does not amplify DNAs from other stud-
ied meats. Quantitative analysis of DNA using qPCR revealed acceptable
precision results. qPCR method can be developed as a standard method
for confirmation of halal meat in meatball products.
Declaration of Competing Interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
CRediT authorship contribution statement
Abdul Rohman: Conceptualization, Methodology, Writing – orig-
inal draft, Funding acquisition. Salmah Orbayinah: Formal analysis,
Writing – original draft. Adam Hermawan: Data curation, Writing –
review & editing. Sismindari Sudjadi: Writing – review & editing, Su-
pervision. Anjar Windarsih: Formal analysis, Writing – review & edit-
ing. Sri Handayani: Formal analysis, Writing – review & editing.
Ethical statement - Studies in Human and Animals
The authors declare that they do not involve humans and do not use
animals in this study.
Acknowledgement
The authors thank to Directorate of Research, Universitas Gadjah
Mada and Pusat Unggulan Perguruan Tinggi Institute for Halal Industri
and Systems (PUI-PT IHIS) 2021 for supporting these research activities.
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