The Sensitivity Evaluation of mt-DNA Genes; NADH Dehydrogenase

Sub Unit 5 (ND5), D-Loop, and Cytochrome-b (Cty-b) to Detect Pork

(Sus scrofa) DNA Isolate and DNA Fragment in Meatball

using PCR Technique

Joni Kusnadi

1,2

and Noval Audi Ashari

Department of Agricultural Product Technology, Faculty of Agricultural Technology, University of Brawijaya,

Jl. Veteran, Malang, Indonesia

Central Laboratory of Life Science, University of Brawijaya Malang, Jl. Veteran, Malang, Indonesia

Keywords: Meatball, mt-DNA, Sensitivity, Primer, Pork.

Abstract: The utilization of mt-DNA primers on previous study specific to detect DNA pork fragments. This study aims

were to evaluate the sensitivity of mt-DNA primers (ND5, D-Loop, and Cyt-b) in pork DNA isolates and its

meatballs product. The sensitivity analysis was conducted in pork DNA isolates with concentrations (10, 1,

-1

, 10

-2

, 10

-3

, 10

-4

, 10

-5

, and 10

-6

ng/µl) and its meatballs product with variation of pork content (0%, 0.01%,

0.05%, 0.1%, 0.5%, 1%, and 5%). Furthermore, the DNA fragment amplification process was carried out

using PCR technique. The amplification results showed that ND5 and Cyt-b primers were more sensitive

because they were able to amplify DNA with concentrations up to 10

-3

ng/µl compared to D-Loop which was

only able to amplify with concentrations up to 10

-2

ng/µl. The sensitivity results using meatballs showed that

ND5 was the most sensitive primer in detecting meatballs with concentration of pork up to 0.01%. It can be

concluded that ND5, Cyt-b, and D-Loop primers are able to detect pork DNA fragment with high sensitivity.

The ND5 primer gave the most sensitive amplification results because it was able to detect pork DNA

fragments with the lowest concentration and meatball with the lowest pork content.

1 INTRODUCTION

There were 1,300 cases of food adulteration between

1980 till 2010 (Moore et al., 2012). Some food

falsification case in various regions in Indonesia are

using pork in resembles beef and applying pork in the

process making of beef meatballs (Bempah, 2017;

Harianmerapi, 2018; Nuryanti, 2020). Meatballs are

food product that often being falsification in the

purpose of economic gain in the anticipation of high

price of beef. Food falsification is defined as an

attempt to replace, imitate, increase, change, or

misrepresent a food product, food packaging, and

fraudulent label information for economic gain

purposes (Hariyadi, 2015).

Identification and prevention of falsification food

is important to protect consumers and prevent

unhealthy competition for food producers especially

processed meat products. Therefore, we need a

detection method that simple and fast for everyday

application (Kesmen et al., 2010). In the analysis of

meatball samples, the meat has undergone high

temperature processing and treatment. Denaturation

of meat protein during heating treatment or some

specific technology applications in the processing

stage may decrease the success of the analytical

method. Therefore, the method is no longer able to

distinguish the species that related closely and

unsuitable for use as an everyday analysis, such as

species-specific detection becomes difficult and long

time (Hofman, 1987; Jemmi and Schlosser, 1992;

Koh et al., 1998; Kesmen et al., 2010).

DNA hybridization and PCR methods have been

widely used for the identification of meat processed

in meat products (Fei et al., 1996; Matsunaga et al.,

1999). Polymerase Chain Reaction (PCR) technique

is widely applied for the analysis of processed meat-

based food products because it is fast, simple,

specific, and sensitive (Matsunaga et al., 1999;

Kesmen et al., 2007; Haunshi et al., 2008; Yahya et

al., 2017). PCR technique is often used as a

technique for fast detection of pork content using

Kusnadi, J. and Ashari, N.

The Sensitivity Evaluation of mt-DNA Genes; NADH Dehydrogenase Sub Unit 5 (ND5), D-Loop, and Cytochrome-b (Cty-b) to Detect Pork (Sus scrofa) DNA Isolate and DNA Fragment in

Meatball using PCR Technique.

DOI: 10.5220/0010507000003108

In Proceedings of the 6th Food Ingredient Asia Conference (6th FiAC 2020) - Food Science, Nutrition and Health, pages 12-19

ISBN: 978-989-758-540-1

mt-DNA primers in the amplification process.

Mitochondrial DNA (mt-DNA) is DNA derived from

mitochondrial organelles with nucleotide that

similar to its parent and lots present in cells (Felk et

al., 2017). Species-specific primers are designed

based on the mt-DNA sequences of various animal

species used in species identification.

The study related to the primer species mt-DNA,

namely NADH dehydrogenase subunit 5 (ND5) with

a target sequence of 227 bp has been used in skeletal

muscle tissue samples from pigs and applied to

detect pork content in typical turkey meat products,

namely Sucuk (Kesmen et al., 2010). Research on

the use of mt-DNA Cyt-b species primers used the

multiplex PCR method to detect the content of pork

which pork had undergone heating treatment at

temperatures of 100°C and 120°C (Matsunaga et al.,

1999). Previous research, mt-DNA primers (ND5, D-

Loop, and Cyt-b) using conventional PCR

techniques applied to beef, goat, pork, lamb, and

chicken have been successfully applied to detect

specific pork content. In this study, mt-DNA primers

(ND5, D-Loop, and Cyt-b) were used to evaluate the

primary sensitivity to various variations of pork

DNA concentrations and the content of pork DNA

fragments in meatballs. This research was conducted

to support the mt-DNA primers specificity data

(ND5, D-Loop, and Cyt-b) in detecting the content

of pig DNA fragments using conventional PCR

techniques and for further research in detecting

cases of counterfeiting food processed products.

2 RESEARCH METHODS

The sensitivity primer test was carried out based on

variations in the DNA concentration of pork and pork

meatballs. The sample of pork DNA isolates had been

obtained and analyzed quantitatively using Nanodrop

(spectrophotometer) produced a DNA concentration

of 132.21 ng/µl and DNA purity of 1.98 (Kusnadi et

al., 2019). Pork DNA isolation samples were diluted

into several concentrations, namely 10 ng/µl, 1 ng/µl,

-1

ng/µl, 10

-2

ng/µl, 10

-3

ng/µl, 10

-4

ng/µl, 10

-5

ng/µl,

and 10

-6

ng/µl. In addition, pork meatballs are made

with a mixture consisting of spices (garlic, onion),

tapioca flour, monosodium glutamate, ice cubes, and

STTP (Sodium tripolipospat). The proportion of each

dough ingredient is shown on Table 1. The producing

of meatballs were mix all ingredients based on

predetermined proportions. The meatball dough was

boiled in water with a temperature of 80°C for 15

minutes until the meatball expands and is cooked. The

meatballs were drained and boiled again at 100°C for

5 minutes, removed, and drained. Furthermore, pork

meatballs with various pork content of 0% (negative

control), 0.01%, 0.05%, 0.1%, 0.5%, 1%, and 5%

(w/w) were DNA isolated using the alkaline-lysis

method with modified procedures.

PCR reactions consist of Go Taq Green Master

Mix (Promega), BSA (Bovine Serum Albumine),

Forward and reverse mt-DNA Primers (ND5, D-

Loop, and Cyt-b), pig DNA with various

concentrations and DNA pork meatballs. The PCR

program consisted of hot start at 95°C for 5 minutes,

denaturation at 95°C for 1 minute, annealing at 54°C

for 1 minute, extension at 72°C for 1 minute, and

post extension at 72°C for 7 minutes. There were 3

mt-DNA primers used, namely ND5, D-Loop, and

Cyt-b with target DNA sequence lengths, namely

227 bp, 835 bp, and 398 bp (Table 2). The

electrophoresis process was used 1.5% agarose gel,

loading dye, 1x TBE, EtBr (Ethidium Bromide), and

1 Kb of DNA ladder. Electrophoresis results were

visualized using gel doc imaging.

Table 1: Meatball ingredient formulation with pork substitution in beef.

Raw Material

Concentration of Beef Substitution with Pork

0% 0.01% 0.05% 0.1% 0.5% 1% 5%

Beef (gr) 40 39.99 39.98 39.96 39.8 39.6 38

Pork (gr) - 0.004 0.02 0.04 0.2 0.4 2

Instant flour for meatballs (gr) 16 16 16 16 16 16 16

Ice Cube (gr) 3.2 3.2 3.2 3.2 3.2 3.2 3.2

STTP (gr) 0.8 0.8 0.8 0.8 0.8 0.8 0.8

Total (gr) 60

The Sensitivity Evaluation of mt-DNA Genes; NADH Dehydrogenase Sub Unit 5 (ND5), D-Loop, and Cytochrome-b (Cty-b) to Detect Pork

(Sus scrofa) DNA Isolate and DNA Fragment in Meatball using PCR Technique

Table 2: Primers used in PCR stages.

Primer Sequence Position (5’- 3’) DNA Length (bp)

ND5 (Kesmen et al.,

2007)

F: 5’-CAT TCG CCT CAC TCA CAT TAA CC-3’

227

R: 5’-AAG AGA GAG TTC TAC GGT CTG TAG-3’

D-loop (Haunshi et al.,

2008)

F:5’-TAC TTC AGG ACC ATC TCA CC-3’

835

R:5’-TAT TCA GAT TGT GGG CGT AT-3’

Cyt-b (Matsunaga et

al., 1999)

F:5’-GAC CTC CCA GCT CCA TCA AAC ATC TCA TCT TGA TGA AA-3’

398

R: 5’-GCT GAT AGT AGA TTT GTG ATG ACC GTA-3’

Figure 1: Sensitivity Test using ND5 Primer in Various Pork DNA. Note: M: DNA ladder 1000 bp, A: initial concentration

(132,05 ng/μl), B: 10 ng/μl, C: 1 ng/μl, D: 10

-1

ng/μl, E: 10

-2

ng/μl, F: 10

-3

ng/μl, G: 10

-4

ng/μl, H: 10

-5

ng/μl, and I: 10

-6

ng/μl.

3 RESULTS

3.1 Sensitivity Test of Primer with

Various Pork DNA Concentrations

The sensitivity analysis aims to determine the primer

ability to detect fragment DNA with the smallest

concentration. In this study, the three species-specific

mt-DNA primers (ND5, D-Loop, and Cyt-b) were

tested at various concentrations of isolated pork

DNA. Previous research results showed that pork

DNA isolates were 132.05 ng/µl with a purity of 1.98

(Kusnadi et al., 2020). The pork DNA isolates has

been used with concentration of 132.05 ng/µl and

purity of 1.98 produced in previous research (Kusnadi

et al., 2020). The amplification results of the three mt-

DNA primers on pork DNA isolates showed that the

lower the DNA concentration used in the

amplification, the thinner the DNA bands produced

(Figures 1, 2, and 3).

Visualization of amplification results using

primers ND5, D-loop, and Cyt-b at various

concentrations of pork were showed that ND5 primers

were able to amplify DNA at DNA concentrations of

10 ng/μl, 1 ng/μl, 10

-1

ng/μl, 10

-2

ng/μl, and 10

-3

ng/μl.

However, the amplification results using the ND5

primer appeared to be thinner than the DNA band

amplified using the Cyt-b primer (Figure 1). Cyt-b

primer was able to amplify DNA with concentration

of 10 ng/μl, 1 ng/μl, 10

-1

ng/μl, 10

-2

ng/μl, and 10

-3

ng/μl constructs with clearer results than D-Loop and

ND5 primers (Figure 3). D-Loop primer was lowest

sensitivity to amplify of DNA samples with

concentrations of 10 ng/μl, 1 ng/μl, 10

-1

ng/μl, 10

-2

ng/μl (Figure 2).

The results of the primer sensitivity analysis have

been carried out before, ND5 primer sensitivity was

able to detect DNA concentrations of 10

-2

ng/µl

(Kesmen et al., 2007), while Cyt-b primer was able to

detect DNA concentrations of 0.25 ng/µl (Matsunaga

et al., 1999), and D-Loop primer has not been studies.

Primers are one of the components that determine the

success of the PCR technique. Primer specificity and

sensitivity analysis are very necessary to support

primary applications to detect contamination in food

processed products.

6th FiAC 2020 - The Food Ingredient Asia Conference (FiAC)

Figure 2: Sensitivity Test using D-Loop Primer in Various Pork DNA. Note: M: DNA ladder, A: initial concentration (132,05

ng/μl), B: 10 ng/μl, C: 1 ng/μl, D: 10

-1

ng/μl, E: 10

-2

ng/μl, F: 10

-3

ng/μl, G: 10

-4

ng/μl, H: 10

-5

ng/μl, dan I: 10

-6

ng/μl.

Figure 3: Sensitivity Test using Cyt-b Primer in Various Pork DNA. Note: M: DNA ladder, A: initial concentration (132,05

ng/μl), B: 10 ng/μl, C: 1 ng/μl, D: 10

-1

ng/μl, E: 10

-2

ng/μl, F: 10

-3

ng/μl, G: 10

-4

ng/μl, H: 10

-5

ng/μl, dan I: 10

-6

ng/μl.

Tabel 3: Concentration and Purity of Pork Meatball DNA Isolate.

Sample

Purity (λ 260/280)

Average

Concentration (ng/μl)

Average

Replicate Replicate

1 2 3 1 2 3

0% 2.12 1.65 1.89 1.89 55.26 142.95 133.05 110.42

0.01% 1.68 1.64 2.01 1.78 26.06 275.82 141.07 147.65

0.05% 2.30 1.92 1.90 2.04 46.75 151.33 90.21 96.0967

0.1% 1.87 1.99 1.93 1.93 63.02 154.03 85.68 100.91

0.5% 2.27 1.87 2.02 2.05 72.90 96.79 128.68 99.4567

1% 2.19 1.86 1.93 1.99 61.85 106.83 83.28 83.9867

5% 2.39 1.87 2.06 2.10 64.60 133.34 144.37 114.103

3.2 Sensitivity Test of Primer in Pork

Meatball Samples

The results of DNA isolation of pork meatballs with

concentrations of pork content of 1%, 0.01%, 0.05%,

0.1%, 0.5%, 1%, and 5% with 3 replications each

resulted in an average concentration of 83.99 to

147.65 ng/µl and the mean purity of 1.78 to 2.10

(Table 3). Based on the DNA concentration that has

been obtained, it shows that the isolation of pork

The Sensitivity Evaluation of mt-DNA Genes; NADH Dehydrogenase Sub Unit 5 (ND5), D-Loop, and Cytochrome-b (Cty-b) to Detect Pork

(Sus scrofa) DNA Isolate and DNA Fragment in Meatball using PCR Technique

meatballs using the alkaline-lysis method produces

high DNA concentrations, however, some samples

still contain RNA and protein contaminants.

Meatball is a processed meat product that has

undergone high temperature treatment and the

addition of various other ingredients. Other

ingredients added to the meatball making, namely

flour, STTP, and salt are added to represent meatball

products on the market. The potential for meatballs to

experience more contamination will be higher and

will result in lowering the concentration of DNA

produced. Based on the results of DNA isolation, it

shows that the alkaline-lysis method is effective for

use as a method for isolating pork meatball DNA, a

slight modification with the addition of Pro-K and

RNAse is needed to reduce protein and RNA

contamination.

The results of DNA isolation of pork meatballs

with pork content of 5%, 1%, 0.5%, and 0.1% were

amplified using three mt-DNA primers ND5, D-Loop,

and Cyt-b primers which were able to produce DNA

bands. D-Loop primer is able to amplify pork

meatballs with pork content of 5% to 0.1% with

different appearance levels, pork meatballs with a

concentration of 5% and 1% can appear for 2

repetitions, while concentrations of 0.05% and 0.01%

appears 1 time. The results of amplification of pork

meatballs with a concentration of 5%, 1%, 0.5%, and

0.1% using Cyt-b primer produced DNA bands with

2 appearances. The amplification results using D-

Loop primer was able to amplify pork meatball

samples at concentrations of 5%, 1%, 0.5%, and

0.1%, however, the resulting DNA bands were not as

good as the amplification results using Cyt-b primer.

The results of DNA isolation of pork meatballs using

ND5 primers were able to amplify DNA up to a

concentration of 5%, 1%, 0.5%, 0.1%, and 0.01%

with an appearance rate of 2 to 3 times. Based on the

results of the primary sensitivity test on meatball

samples, the ND5 primer was the most sensitive

compared to Cyt-b and D-Loop, it was shown that the

primer was able to amplify meatball samples with the

lowest pork concentration of 0.01%. Pork samples

have undergone a processing process (milling and

heating) and it is possible to experience degradation

which can reduce the quality of the sample DNA. The

results showed that the meatball sample with the

lowest concentration of pork DNA content of 0.01%

could be amplified using ND5 primer. The

amplification results using ND5 primer produced 227

bp of target DNA strands. Compared to D-Loop and

Cyt-b primers, ND5 primer produced the shortest

length of the target DNA sequence. The results

showed that primers with a short target DNA length

such as ND5 with 227 bp length to detect specific

species in preheated foods gave the most sensitive

results. The results of amplification of pork meatballs

with D-Loop primer were able to detect the content of

pork meatballs with a concentration of 5% to 0.1% in

meatballs. The D-Loop primer has a target DNA

sequence length of 835 bp (Figure 5). Amplification

using D-Loop primers results in lower sensitivity

compared to ND5 primers.

The amplification results using Cyt-b primer

showed that the primer was able to detect the content

of pork with concentration of 5% to 0.1% in

meatballs. The Cyt-b primer has a target DNA

sequence with length of 398 bp. Based on the results

of the sensitivity analysis, it shows that D-Loop and

Cyt-b primers are sensitive in detecting pork content

Figure 4: Sensitivity Test Using ND5 Primer in Pork Meatballs. Note: A. Pork Meat Concentration 5%, B. Pork Meat

Concentration 1%, C. Pork Concentration 0.5%, D. Pork Concentration 0.1%, E. Pork Meat Concentration 0.05%, F. Pork

Concentration 0.01%, G. Pork Concentration 0%, (-) Negative Control, (+) Positive Control, M. DNA Ladder.

6th FiAC 2020 - The Food Ingredient Asia Conference (FiAC)

Figure 5: Sensitivity Test using D-Loop Primer in Pork Meatballs. Note: A. Pork Concentration 5%, B. Pork Meat

Concentration 1%, C. Pork Concentration 0.5%, D. Pork Concentration 0.1%, E. Pork Meat Concentration 0.05%, F. Pork

Concentration 0.1%, G. Pork Concentration 0%, (-) Negative Control, (+) Positive Control, M. DNA Ladder.

Figure 6: Sensitivity Test Using Cyt-b Primer in Pork Meatballs. Note: A. Pork Concentration 5%, B. Pork Meat

Concentration 1%, C. Pork Meat Concentration 0.5%, D. Pork Meat Concentration 0.1%, E. Pork Meat Concentration 0.05%,

F. Pork Concentration 0.1%, G. Pork Concentration 0%, (-) Negative Control, (+) Positive Control, M. DNA Ladder.

with concentration of rok content 5% to 0.1% in

meatballs. The overall results of primer sensitivity

analysis showed that the ND5 primer was the most

sensitive in detecting pork content in meatballs

compared to D-Loop and Cyt-b primers. ND5 primers

had the shortest target gene with sequence length of

227 bp compared to both D-Loop and Cyt-b primers,

this was thought to cause the primer to have the

highest sensitivity.

In this study, the results of the ND5 primer

sensitivity test showed the most sensitive primer was

able to detect up to 0.01% pork concentration in

meatballs. Previous research results of ND5 primer

had low sensitivity in detecting pork content, namely

a concentration of 0.1% in sausage samples (Kesmen,

2007). Meanwhile, in another study, the ND5 primer

had low sensitivity and was able to detect the content

of pork with a concentration of 0.1% in a sample of a

mixture of chicken meat and lard (Felk et al., 2017).

In this study, Cyt-b primer had lower sensitivity

compared to ND5 primer. Another study, Cyt-b

primer had low sensitivity in detecting pork with a

concentration of 1% in a mixture of cooked pork and

beef.

The high sensitivity of the ND5 primer can be

caused by the reason that the orimer has a short DNA

sequence target so that it is sensitive in detecting

meatball samples. PCR testing uses a primer with a

short amplicon target which aims to detect specific

species in foods given heat treatment, is better and

more stable than using primers with a long amplicon

target (Ali et al., 2016). This is supported by Rahman

et al (2014), that primers with short amplicon targets

can be amplified with samples processed by high

pressure autoclave. Yoshida et al. (2009) who tested

primers with a target of 126 bp and 83 bp with their

samples given heating and pressure treatments, also

showed that primers with shorter amplicon targets

were more sensitive for detecting DNA fragments.

The Sensitivity Evaluation of mt-DNA Genes; NADH Dehydrogenase Sub Unit 5 (ND5), D-Loop, and Cytochrome-b (Cty-b) to Detect Pork

(Sus scrofa) DNA Isolate and DNA Fragment in Meatball using PCR Technique

The presence of a mixture of flour, spices, salt,

and STTP can potentially be a contaminant in DNA

isolates. The presence of these contaminants can

interfere with the success of the PCR process and are

not repeatable. The results showed that the

occurrence rate of DNA bands from 3 repetitions

varied. The high content of polysaccharides in

isolates originating from flour, contamination of

organic compounds such as polysaccharides may

interfere with the enzymatic process of DNA

polymerase by mimicking the structure of nucleic

acids (Schrader et al., 2012). According to Bergallo

et al (2006), there are several ways to remove these

contaminants, for example, such as giving Tween 20

to remove polysaccharides.

4 CONCLUSION

The mt-DNA ND5 and Cyt-b primers were more

sensitive because they were able to amplify DNA up

to concentration of 10

-3

ng/µl compared to D-Loop

primer which were only able to amplify at

concentrations up to 10

-2

ng/µl. In addition, ND5

primer was the most sensitive to detect meatballs with

pork content up to 0.01%. Thus, the three mt-DNA

ND5, Cyt-b, and D-Loop primers were able to detect

specific and sensitive pork DNA fragments. The ND5

primer gave the most sensitive amplification results

because it was able to detect pig DNA fragments with

the lowest concentration of 10

-3

ng/µl and meatballs

with the lowest pork content of 0.01%.

ACKNOWLEDGEMENTS

Thank you very much to Brawijaya University for

supporting research funding through the Doctoral

Grant Program in 2019 and 2020. Thanks also to the

Central Laboratory of Life Sciences (LSIH) which

has facilitated the implementation of research.

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