Detection of Rat (Rattus Norvegicus) DNA Fragments using Specific:

Species Primer mt-DNA 12S rRNA and Cyt-b with Polymerase Chain

Reaction (PCR) Technique

Ersita Putri Aisah

and Joni Kusnadi

1,2

Department of Agricultural Product Technology, Faculty of Agricultural Technology,

University of Brawijawa Malang, Indonesia

Central Laboratory of Science, University of Brawijaya Malang, Jl. Veteran, Malang 65145, Indonesia

Keyword: 12S rRNA, Mitochondrial DNA, PCR, Primer.

Abstract: A pair of species-specific mt-DNA primer 12S rRNA has been designed based on rat (Rattus norvegicus)

DNA sequences. In this study, the specificity test of 12S rRNA and Cyt-b were carried out to detect rat DNA

fragments. Samples of this research were used non-halal animal meat consisted of rats, lamb, dog, pig and

halal animal meat consisted of cow, chicken, and horse. Furthermore, DNA was isolated from animal meat

using modification of chloroform isoamyl alcohol method then quantitatively tested for its concentration and

purity. Animal meat isolates were amplified using 12S rRNA and Cyt-b primers using PCR techniques.

Analysis of PCR results using agarose gel electrophoresis 1.5%. The amplification results showed that 12S

rRNA primer produced DNA bands of 228 bp length and Cyt-b primer produced DNA bands of 603 bp length.

The amplification results showed that both of 12S rRNA and Cyt-b primers were specific to detect rat DNA

fragments. Thus, both of primers are recommended to be further tested for sensitivity and applied to processed

meat products such as meatballs, sausages, and corned beef.

1 INTRODUCTION

Rats often cause health issues such as bubonic plague,

leptospirosis, murine typhus, and plague (Center for

Disease Control and Prevention, 2011). Ironically, rat

meat holds a high possibility to be used in various

food products, most of the time, meatballs. This

adulteration case rises among society in Indonesia as

it exhibits promising profit. Moreover, that such

adulteration could not be easily identified. The recent

case of meat adulteration were 63 tons. Those cases

were substituted beef into pig meat that claimed as

pure beef (Warta Ekonomi, 2012). This criminal case

complicate the Muslims society as they are prohibited

from consuming non-halal foods. The lack of food

management that related to halal, safety, and health

resulting the production process violations (Yasmin,

2013). The low awareness of the importance about

halal logo, halal certification at Slaughterhouses

(RPH) and Poultry Slaughterhouses (RPU), also the

low the protection from local governments leads the

increasing of new cases arise (Arifiani, 2019).

Indonesian Law no. 33 year 2014 regarding to Halal

Product Guarantee is regulated to eliminate the meat

adulteration cases.

The developing of an effective and efficient

method in meat contamination detection and

adulteration are essential in order to support the

guarantee of Halal. Polymerase Chain Reaction

(PCR) is a method of DNA analysis by amplifying

DNA in vitro involving several stages, it’s

denaturation, annealing, and extension (Handoyo et

al., 2000). Some constituent components including

DNA template (DNA template); primer;

deoxynucleoside triphosphates (dNTPs); DNA

polymerase enzyme; and PCR buffer, are required to

optimize the process. The primers were analyze as

primers are the success key in PCR technique. Mt-

DNA 12S rRNA gene contains high base variations

between species and low base variations in the same

species (Kitpipit, 2014). Several advantages of the

PCR technique are its specificity and high sensitivity,

short time, and its ability to detect contaminated

samples and to work on samples with a complex

mixture (Aminah et al., 2019). The PCR technique is

known as accurate, fast, affordable, and able to

Aisah, E. and Kusnadi, J.

Detection of Rat (Rattus Norvegicus) DNA Fragments using Speciﬁc: Species Primer mt-DNA 12S rRNA and Cyt-b with Polymerase Chain Reaction (PCR) Technique.

DOI: 10.5220/0010507100003108

In Proceedings of the 6th Food Ingredient Asia Conference (6th FiAC 2020) - Food Science, Nutrition and Health, pages 20-24

ISBN: 978-989-758-540-1

analyze a DNA (Deoxyribonucleic acid) sample from

high to low concentration (Arslan et al., 2006;

Aminah et al., 2019).

Specific primers of mt-DNA 12S ribosomal RNA

have been developed for detecting and identifying

DNA fragments of cats (Felis catus), dogs (Canis

familiaris), and rat (Rattus norvegicus) that contained

in food and food ingredients. The results showed that

3 pairs of primers that had been designed from the

DNA of 24 animals and plants produced specific

sequences with lengths of 108 bp, 101 bp, and 96 bp

were successfully amplified in cats, dogs, and rat

(Martin et al., 2014). The comparison primer was mt-

DNA Cyt-b which had been tested for it’s specificity

on detected DNA fragments of rat (Nuraini et al.,

2012). Cyt-b primers that designed manually, had

used to amplify 6 species which are goats, chickens,

cows, lamb, pigs, and horses successfully, while the

Cyt-b primers that designed using software based on

the Cyt-b sequence on Rattus norvegicus were

success to amplify 7 animal species which indicated

that the Cyt-b primer was specific. It is possible to

detect tissue samples of cats, dogs, rats on food and

foodstuffs in high sensitivity and specificity by using

specific primers (Martin et al., 2014). In this study, a

species-specific mt-DNA 12S rRNA primer was

designed and the specificity was analyzed in detecting

DNA fragments in rats as the initial stage of testing.

The comparison genes used mt-DNA Cyt-b primers

that had been studied previously for the detection of

rat DNA fragments. It is expected that further

research will be develop in concerning primer testing

based on the primer sensitivity and its applicate on

detecting rat DNA fragments in various meat

products such as meatballs, corned beef, and

sausages.

2 RESEARCH METHODS

The samples of non-halal raw meats were collected

from rats (positive control), dogs, and pigs while the

halal meat samples were obtained from cattle,

chicken, horse, and lamb (negative control). The

DNA isolation was performed by Chloroform

Isoamyl Alcohol method modified from Sambrook

and Russel’s (1989) procedure. The reagent used in

DNA isolation were consisted of STE lysis buffer (0.1

M NaCl, 1 mM EDTA, 10 mM Tris-HCl pH 8), 10%

SDS, and 10 mg / mL Proteinase-K. C: I (Chloroform:

Isoamyl) with a ratio of 24:1 was used in the

purification stage. Absolute ethanol (EtOH) was used

in the DNA washing stage, treated on cold condition.

In the DNA precipitation stage, 70% ethanol and 5M

NaCl were used. The final stage was DNA elution

that used TE buffer pH 7.6. The modified procedures

were used Pro-K instead of phenol in the DNA

purification stage.

The results of DNA isolation were analyzed

quantitatively using (Nanodrop / ND-1000 UV / Vis)

to obtain the concentration and purity, and were

amplified using Thermal Cycler (Applied Biosystem

/ PCR System 9700) and Thermocycler (Takara /

Version 3 Model TP600). The PCR process with a

total volume of 10µl consisted of 5 µl Go Taq Green

Master Mix (PROMEGA), a pair of 12S rRNA

primers with a concentration of 5 pmol / µl as much

as 0.5 µl, DNA template 1 µl, and ddH2O 3 µl. The

PCR program for primers 12S rRNA consisted of five

stages. Firstly, the initial denaturation at 95˚C for 5

minutes. Secondly, denaturation at 95˚C for 30

seconds. Thirdly, the 12S rRNA primers annealing at

54˚C for 45 seconds. Fourth, the extension at 72˚C for

30 seconds, and the last, final extension with a

temperature of 72˚C for 5 minutes. The same program

was applied for Cyt-b primers amplification,

excluding the annealing step 54˚C for 45 seconds.

12S rRNA gene primers were specifically

designed from mt-DNA rat (Rattus norvegicus) based

on the data from GenBank NCBI (National Center for

Biotechnology Information). The primers were

designed manually by bioinformatics programs such

as clustal X and bioedit to align the 12S rRNA gene

sequences from several test animals, such as cattle

(Bos indicus), dogs (Canis lupus familiaris), pigs (Sus

scrofa domesticus), chickens (Gallus gallus), horse

(Equus caballus), and lamb (Ovis aries). This study

was performed the 12S rRNA gene with a target

length of 228 bp employing a primer sequence length

of 20 bp, GC content (50%), melting temperature of

60°C, and annealing temperature of 54 °C (Figure 1).

Table 1: Primers 12S rRNA and Cyt-b genes.

Primer Sequence (5’ – 3’) Amplicon (bp) Base

12S rRNA

Forward: GGA CCT

AAG CCC AAT AAC

228

Reverse: TTC TAC

CTT ACC CCT TCT

Cyt-b

Forward: GAC CTC

CCA GCT CCA TCA

AAC ATC TCA TCT

TGA TGA AA

603

Reverse: GAA TGG

GAT TTT GTC TGC

GTT GGA GTT T

The comparison primers used forward SIM

primers which were designed based on the Cyt-b gene

Detection of Rat (Rattus Norvegicus) DNA Fragments using Speciﬁc: Species Primer mt-DNA 12S rRNA and Cyt-b with Polymerase Chain

Reaction (PCR) Technique

sequence from 6 animal species. Forward SIM

primers were selected according to sequence

missmatch within control species of 3-5 bases for 38

bp. SIM was designed longer than species-specific

primers with nucleotides measuring 26-29. DNA

fragments were formulated using software for

determining primers that were designed based on

species-specific areas. Reverse primers for detecting

the DNA fragments of rat were designed based on

primer design software

(http://www.ncbi.nlm.nih.gov/tools/primerblast/inde

xshtml). Reverse primers for rat DNA fragments

detection have a target sequence of 603 bp. (Nuraini

et al., 2012).

The amplification results were qualitative

analyzed using 1.5% agarose gel electrophoresis

(agarose, TBE buffer, EtBr, loading dye, and 1 kB

DNA ladders). The electrophoresis was performed

using Horizontal Electrophoresis (Mupid 2 Plus) with

a power of 50 V for 45 - 55 minutes and visualized

using Chemidoc Gel Imaging (Bio-Rad / BR-200).

Table 2: Concentration and Purity of Fresh Meat DNA

Isolate.

Sample Purity (λ 260/280) Concentration (ng/µl)

Rat 1.98 152.19

Mice 1.94 62.98

Chicken 1.72 90.20

Cattle 1.50 111.16

Horse 1.72 77.33

Lamb 1.96 392.78

Dog 1.84 50.31

Pig 1.96 60.30

3 RESULT AND DISCUSSION

Based on the results, DNA isolation performing

modified Chloroform Isoamyl Alcohol (PCI) method

on fresh meat samples from various non-halal animals

produced DNA with concentrations ranging from

50.30 ng / µl to 392.78 ng / µl with a purity of 1.50 to

1.98 (Table 2). The highest DNA concentration was

found in lamb meat while the lowest was in cattle.

The protein contaminants with purity values of 1.50,

1.72, and 1.72 were detected in most of the pure DNA

isolations, except in cattle, horse, and chicken. The

purity values with less than 1.8 indicate, there is a

presence of protein contamination, while the values

more than 2.0 indicate that there’s a presence of RNA

contamination (Nzilibili et al., 2018). The presence of

protein contamination in DNA isolation results

implies a shortage of effectiveness in using Pro-K to

denature all proteins in chicken, horse, and cattle

meat. In the future study, a modified with the addition

of Pro-K or phenol needs to be done in order to

maximize the protein denaturation on the purification

stage. The PCI method using proteinase on DNA

isolation was more effective comparing the used of

ammonium to remove the protein content

(Minematsu et al., 2004; Haunshi et al., 2008).

The DNA isolation was performed based on

comparing the PCI conventional method and

commercial KIT to produce a high-quality DNA

isolation. However, there are several inadequacy on

PCI method which required a longer time and toxic

reagents content: phenol, chloroform, SDS, etc.

Besides, the use of commercial KIT is relatively

expensive. Modification of the PCI method has been

used to produce good quality DNA isolates without

smear even though it was used only one purification

stage (PCI and incubation with minimal time)

(Haunshi et al., 2008).

The specificity test was used the DNA isolates

from several animals and amplify used PCR

technique. The amplification results that used 12S

rRNA primers showed DNA bands in rat meat

samples with sizes ranging from 228 bp.

Figure 1: The site of the design of the primer attached to the 12S rRNA sequence Rattus norvegicus, note: yellow is forward

primer and red is reverse primer.

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

The DNA band was appeared in rat samples which

indicated the primers are specific to amplify the 12

rRNA gene sequence on rat (Figure 1). While, the

amplification result used Cyt-b primers as the

comparison gene was formed DNA bands with 603

bp in the rat meat sample. This result was verified that

both 12s rRNA and Cyt-b primers are specific to

amplify rat DNA sequences (Figure 2). Cyt-b primers

are often used to compare the phylogenetics of

species in the same genus or the same family. Several

studies of the genetic diversity of the Cyt-b gene have

been studied in cattle (Bos taurus), lamb (Ovis aries),

and goats (Capra hircus), roebuck (Capreolus

capreolus), and red deer (Cervus elaphus) (Wolf et

al., 1999; Nuraini et al., 2012). Rats (Rattus

norvegicus) have a long Cyt-b sequence of 1143 bp

(Nuraini et al., 2012).

Figure 2: Result of 12S rRNA Primer. Note: E1: (-), E2:

Rat, E3: Mice, E4: Chichken, E5: Cattle, E6: Horses, E7:

Lamb, E8: Dog, E9: Pig, M: DNA Marker 100 bp.

The success amplifying uses PCR technique was

demonstrated by the 12S rRNA and Cyt-b primers

tested on various animal meat isolates producing

target DNA fragments with different lengths

according to the length of the targeted DNA. Cyt-b

primer has been used previously to detect rat DNA

fragments resulting in 603 bp of target DNA (Nuraini

et al., 2012) which conforms to the results obtained in

this study.

The result of the 12S rRNA primer specificity test

showed that the amplified band was formed in the

meat sample of rats with a length of 228 bp. It shows

that the mitochondrial DNA of the 12S rRNA gene

can be used as a marker or primer for specific species

to identify species as it has a large variation among

the species (Springer and Douzery, 1996), and 12S

rRNA is mostly used in intra and inter-species

phylogenetic studies (Tougard et al., 2001). In the

other hand, the examined of specificity of Cyt-b

primer were represent the quite clear bands with 603

bp of rat meat sample. The Cyt-B primer used in this

study had quite specific properties (Nuraini, 2012).

The Cyt-b primer was further examined on various

samples contained some mixtures beef and pork or

pork and rats with varying concentrations (1%, 5%,

10%, 15%, 20%, and 25%). As a result, the specificity

of the Cyt-b primer was shown by a specific primer

detecting samples of rat DNA fragments contained in

a mixture of beef and rat. Meatballs which are

composed of 15% rat meat would be more clearly

detected compared to 1% that could not be detected

due to the very small value (Nuraini et al., 2012). The

succession of primers to amplify the DNA target was

supported by the primers sequence contain of

nucleotide bases that might specifically hybridized to

DNA template (Yuwono, 2006) and a nucleotides

sequence originating from the DNA target. A good

primer was consisted of nucleotide bases that

conserved on the template, thus not exist in any other

location on its template (Pelt-verkuil et al., 2008). The

succession of specifically designed of 12S rRNA

primers are need further analyzed to detect the

sensitivity on rat DNA fragments (Rattus

norvegicus). In addition, the 12S rRNA primer needs

to be applied in detecting rat DNA fragments in

various processed food products. Thus, conventional

PCR techniques using species-specific primers are

qualifiable to detect DNA fragments of non-halal

animals (Rattus norvegicus) with specifically,

rapidly, and conveniently.

4 CONCLUSION

Based on the results in this study, the 12S rRNA

primer specificity test on rat meat samples produced

a sequence with 228 bp. Therefore, both 12S rRNA

and Cyt-b are specific primers which able detect rat

DNA fragments (Rattus norvegicus). Nevertheless,

there is still need to undertake further examine on

specific 12S rRNA regarding to the sensitivity of the

primers in detecting rat DNA fragments to obtain the

LOD (limit of detection) sample concentration and its

application in various meat products.

Figure 3: Result of Cyt-b primer amplification. Note: G1: (-

), G2: Rat, G3: Mice, G4: Chicken, G5: Cattle, G6: Horses,

G7: Lamb, G8: Dog, G9: Pig, M: DNA Marker 100 bp.

Detection of Rat (Rattus Norvegicus) DNA Fragments using Speciﬁc: Species Primer mt-DNA 12S rRNA and Cyt-b with Polymerase Chain

Reaction (PCR) Technique

ACKNOWLEDGEMENTS

The author would like to thank Brawijaya University

for funding this research through the 2020 Doctoral

Grants Program. I would also like to express my

gratitude to the LSIH Laboratory for facilitating

various equipment to support the continuity of the

research.

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