Article Review: Testing for Detection of Low Pig (Porcine) DNA in Cosmetic Products and Health Supplements

Raden Indah Puspita Syafitri; Naqiyyah Fil Ayatil Karimah; Soraya Ratnawulan Mita

About The Authors

Raden Indah Puspita Syafitri
Pharmacy Professional Program, Faculty of Pharmacy, Universitas Padjadjaran
Indonesia

Pharmacy Professional Program, Faculty of Pharmacy

Naqiyyah Fil Ayatil Karimah
Pharmacy Professional Program, School of Pharmacy, Institut Teknologi Bandung
Indonesia

Pharmacy Professional Program

Soraya Ratnawulan Mita
Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Universitas Padjadjaran
Vanuatu

Department of Pharmaceutics and Pharmaceutical Technology

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Article Review: Testing for Detection of Low Pig (Porcine) DNA in Cosmetic

Products and Health Supplements

Raden Indah Puspita Syafitri1*, Naqiyyah Fil Ayatil Karimah2, Soraya Ratnawulan Mita3

1 Pharmacy Professional Program, Faculty of Pharmacy, Universitas Padjadjaran

2 Pharmacy Professional Program, School of Pharmacy, Institut Teknologi Bandung

3Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy,

Universitas Padjadjaran

e-mail: radenindah16@gmail.com

Submitted: 05/06/2023, Revised: 06/06/2023, Accepted: 16/08/2023, Published: 17/04/2024

Abstract

The increase in the Muslim population worldwide has led to rapid growth in the

halal product industry. Currently, halal food holds the largest proportion in the

global market for halal products, followed by cosmetics and health supplements as

one of the main sectors. In the raw materials used for capsule shells and the

manufacture of cosmetics, many ingredients come from non-halal sources, such as

gelatin, fatty acids, glycerin, and collagen, which are derived from pigs. However,

the presence of pork ingredients in cosmetic products and health supplements is

prohibited in certain religions, such as Islam. Therefore, the detection of lard in

cosmetic and pharmaceutical products plays a crucial role for the benefit of

Muslim consumers. Many methods have been developed to detect pork content in

highly processed products, one of which is the DNA-based method. This literature

review was conducted to explain DNA isolation methods and detection techniques

that can be used for pig DNA analysis. A literature search was performed through

online databases, and 14 research journals were found that met the inclusion

criteria. Based on the collected research, DNA-based methods, including the

Polymerase Chain Reaction (PCR) and its modifications, have demonstrated the

ability to detect porcine DNA in products like cosmetics and health supplements.

The selection of DNA detection and isolation methods depends on the type of

Vol. 5, Issue 2, 2023 (385-404)

http://journal.unpad.ac.id/IdJP

*Corresponding author,

e-mail: radenindah16@gmail.com (R. I. P. Syafitri )

https://doi.org/10.24198/idjp. v5i2.47259

386

1. Introduction

The global halal product market

has seen rapid growth, driven by the

increasing Muslim population. Halal food

dominates the market, followed by

cosmetics and pharmaceutical products as

key sectors (1). In Islam, a product is

considered non-halal if it contains

ingredients derived from pigs, dogs,

animal blood, alcohol, and other animals

that are not slaughtered in accordance

with Islamic guidelines such as pigs,

carnivorous animals (lions, tigers,

leopards), birds of prey (eagles, hawks,

vultures, and falcons), insect and animals

found dead. (2). Several Islamic

countries such as Indonesia have

established strict regulations for

producers and importers in product labels,

which is halal certificates in order to

distinguish them from non-halal products

(3). Halal certification is required so that

a product can enter the halal market. Each

product must go through a rigorous

qualification process to ensure that it does

not contain haram elements (4).

Gelatin is one of the raw

materials that is often used in the

manufacture of soft capsule shells for

medicinal products and health

supplements. Gelatin is a very popular

ingredient used in various food and

pharmaceutical products (5). In large-

scale gelatin manufacture, generally the

main raw material used is collagen found

in cattle and pigs (6). In some cosmetic

products, ingredients derived from pigs

such as fatty acids, glycerin and collagen

can be used in the production of body

lotions, creams and masks (7). In most

countries, lard substituents in cosmetic

formulations are commonly used as an

emulsifying agent, and viscosity

increasing agent (8). However, the

detection of lard in cosmetic products and

other pharmaceutical products plays an

important role for the benefit of Muslim

consumers.

Many methods have been

developed to detect pig content in highly

processed products, including

physicochemical properties-based

methods, and DNA based methods.

R. I. P. Syafitri et al / Indo J Pharm 5 (2023) 385-405

sample under examination, thus enhancing the accuracy and success of porcine

DNA detection.

Keywords: DNA analysis, Pig DNA, Pig Content, Cosmetics, PCR, Health

Supplements.

387

One of the physicochemical

properties-based methods that can be used

is Fourier transform infrared spectroscopy

(FTIR). However, the FTIR method has

limitations in detecting pork content due

to low sensitivity, sample purity

requirements, and the inability to

differentiate gelatin mixtures from

different sources due to similar structures

and properties (9–11).

Protein/antigen-based methods

such as high-performance liquid

chromatography (HPLC) and Enzyme-

Linked Immunosorbent Assay (ELISA)

can also be used to detect pig content.

However, the HPLC method has

limitations in distinguishing gelatin

mixtures due to similar chemical

properties (9). Meanwhile, antigen-

antibody interaction-based methods like

ELISA have limitations, including cross-

reactions, low sensitivity, false positives

at low ionic strengths, and interactions in

the presence of inhibitors (9,11,12).

DNA-based detection methods for

pork content in highly processed products

are known to be specific, reproducible,

sensitive, have fast processing times and

require low costs (13). DNA is unique and

specific to each species, allowing for

accurate detection of pork content. Its

uniqueness and specificity enable

differentiation between species and

individuals, making it a reliable method

for detecting pork in various products

(14). Polymerase Chain Reaction (PCR)

is a method that can be used to detect pig

DNA in cosmetics and health

supplements.

Detecting porcine DNA in cosmetics and

health supplements is challenging due to

the high processing involved, which leads

to DNA degradation into short fragments.

This fragmented DNA poses difficulties

in detection (12,15). In addition, some of

the excipients used in health supplements

can absorb DNA, making the detection of

DNA in capsule shells made of gelatin

more difficult (9). This literature review

focuses on methods for isolating and

detecting DNA in processed products,

such as gelatin capsule shells and

cosmetics, to optimize the detection of

porcine DNA. It provides insights into

various DNA isolation and detection

methods, as well as their detection limits

for porcine DNA analysis.

2. Method

Literature searches were

conducted using keywords such as DNA,

R. I. P. Syafitri et al / Indo J Pharm 5 (2023) 385-405

388

porcine, dietary supplement, cosmetics,

gelatin, and halal on online databases

including Google Scholar and PubMed.

The reference journals for this review are

from the last 10 years (2013 – 2023). The

inclusion criteria are journals regarding

testing for the detection of porcine DNA

in samples of health and cosmetic

supplements. Exclusion criteria involve

journals with incomplete or restricted

access to relevant data.

R. I. P. Syafitri et al / Indo J Pharm 5 (2023) 385-405

Tabel 1. Methods performed for review.

Basic Data Literatur Search:

Google Scholar and Pubmed

Search result

(n = 191)

Article after duplicates

removed

(n = 180)

Filtered articles based

on whole text.

Article included in the

analysis

(n = 14)

Search result that will be

removed

(n = 166)

389

3. Result

Based on literature searches, data

were obtained from several journals that

tested pig DNA on cosmetic samples and

health supplements using the Polymerase

Chain Reaction (PCR) detection method

with different DNA extraction methods as

well as primers and gene targets. There

are 14 journals used for this literature

review which are summarized in the

following Table 2 and Table 3.

R. I. P. Syafitri et al / Indo J Pharm 5 (2023) 385-405

Table 2. Scientific Journal Data related to DNA Analysis in Cosmetics and Health

Supplements (Gelatin)

Test

Sample Analysis Method Primers

and Gene

Probes /Targets Detection

Limit References

Cosmetics

(Cream)

Detection

method:

PCR

MyTaq

DNA

Polymerase

DNA

Extraction Method:

Wizard

Genomic

DNA

Purification

Kit

Primer

SP and 12

SFW

(Porcine

Specific)

from

Mt-DNA

Gene

targets:

S rRNA

.001ng/µL (16)

Cosmetics

(Cream)

Detection

method:

Real

-time

Polymerase

Chain

Reaction

DNA

Extraction Method:

CATB,

Power

PrepTM

DNA

extraction

from

food

and feed

kit

(KogeneBiotech),

QIAamp

DNA stool

mini

kit

(Qiagen,

Hilden,

Germany),

TIANamp

Genomic

DNA

kit

(Tiangen

Biotech,

Beijing,

China),

and

Wizard

Genomic

DNA

purification

kit

(Promega,

Madison,

WI,

USA)]

Primer

•

Sus

(Porcine

Specific)

from Mt

DNA

•

Sus NDH

(Porcine

Specific)

from Mt

DNA

Gene

targets:

ndh

Liquid

preparation

.28 x 100

copies

Powder

dosage

: 2.

101 copies

Cream

preparation

.28 x 100

*Threshold

cycle

(Ct)

(7)

390

R. I. P. Syafitri et al / Indo J Pharm 5 (2023) 385-405

Cosmetics

(Liquid,

powder

and

cream

preparations)

Detection

Method:

TaqMan

Probe real-

time

Polymerase

Chain

Reaction

DNA

Extraction Method:

CATB

Primer

•

VIC /MGB

probe,

limited

primer

from

Mt-DNA

•

The

primer

used for the

pig

species

was

the

Meat

Animal

Research

Centre

Porcine

Repetitive

Element

Gene

targets:

•

18s rRNA

gene

(Universal)

•

Specific

primers target

multiple

porcine

repetitive

element

(MPRE) genes

Undetectable

(17)

Gelatin

Capsule

Shell

Detection

Method:

PCR

-RFLP and

Duplex

PCR

. PCR-RFLP

Extraction

Method:

DNeasy

Mericon Food

Kit

(Qiagen,

Germany)

Primer

Oligonucleotide

which

contains

universal

primer

and

porcine-

specific

primers

Restriction

Enzyme:

BsaJI

.01% DNA

.01 gram/

100

DNA

(18)

391

R. I. P. Syafitri et al / Indo J Pharm 5 (2023) 385-405

Gelatin

and

Gelatin

Capsules

Detection

method:

Real

-time

Polymerase

Chain

Reaction

DNA

Extraction Method:

MasterPure

DNA

Purification

Kit

Primer

•

16S

(Universal)

from Mt-DNA

•

CBH

(Porcine

Specific)

from

Mt-DNA

•

MPRE

(Porcine

Specific)

from

Nuclear DNA

Gene

Targets

•

16S

rRNA

(Universal)

•

Cyt-b

(Porcine

Specific)

•

MPRE41

(Porcine

Specific)

Nuclear

DNA

pg of

DNA

gelatin

Mitochondrial

DNA

gelatin

(19)

Gelatin

capsule shell

Detection method:

PCR

-RFLP

DNA Extraction Method:

FavorPrep Food DNA

Extraction Kit (Favorgen

Biotech Corp, Ping

-Tung,

Taiwan)

Primer:

•

Eukaryotic

primer

(Universal)

from Mt-DNA

•

Porcine

primer

(Porcine

Specific) from

Mt-DNA

Restriction Enzymes:

•

BsaAI

Gene targets:

•

18S rRNA

(Universal)

•

Cyt-

B (Porcine

Specific)

0.001 ng of pig

DNA

(20)

392

R. I. P. Syafitri et al / Indo J Pharm 5 (2023) 385-405

Health

Supplements

Detection Method:

Taqman Qualitative PCR

Multiplex Probe

DNA Extraction Method:

FavorPrep Food DNA

Extraction Kit (Favorgen

Biotech Copr, Ping

Tung,

Taiwan)

Primer:

•Eukaryotic 18S

rRNA

(Universal)

from Mt-

DNA

•Porcine primer

Mt-DNA

Porcine

specific)

Gene targets:

•

18S rRNA

(Universal)

•

Cyt-

B (Porcine

Specific)

0.005 ng/µL

DNA in

gelatin

mixture

(21)

Gelatin Raw

Materials

Detection Method:

qPCR

DNA Extraction Method:

NucleoSpin® Food

(Macherey

-Nagel GmbH

& Co. KG, Postfach,

Germany), DNeasy

Mericon Food Kit (Qiagen

GmbH, Hilden, Germany),

QuickGene DNA Tissue

Kit L (Kurabo Industries

ltd., Osaka, Japan), and

Foodproof® GMO Sample

Preparation Kit (Biotecon

Diagnostics GmbH,

Postdam, Germany).

Primer:

•

Universal

primers used

for mammalian

species were

researcher-

designed.

•

Primers used

for the pig

species were

researcher-

designed

Gene Targets:

•

Universal

primers target

the GH

(growth

hormone) gene

•

Specific

primers target

prion protein

genes

pg/µL (11)

393

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Gelatin

Capsule

Shell

Detection Method:

Conventional PCR

DNA Extraction Method:

DNeasy

mericon

Food Kit

(Qiagen, Hilden,

Germany)

Primer:

Porcine specific

primer

used was

AF039170

Gene Targets:

Cyt

b gene (porcine

specific)

0.1% w/w

(15)

Gelatin

Capsule

Shell

Detection Method:

PCR

DNA Extraction Method:

MasterPure

DNA

Extraction Kit (

Epicentre

Madison, WI, USA) with

pH optimization

Primer:

Porcine specific

primer

used is as

follows.

•

SUS_FWD &

SUS RVS

•

SWF & SWR

•

SWF &

SUSC2R

•

SUS_FWD &

SUSN3R

Gene Targets:

•

SUS_FWD &

SUS RVS:

ND5

•SWF & SWR:

Cyt-b

•

SWF &

SUSC2R: Cyt

•

SUS_FWD &

SUSN3R: ND5

0.1 pg

(12)

394

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Gelatin

powder &

gelatin

capsule

shells

Detection Method:

PCR with Loop

Mediated

Isothermal Amplification

(LAMP)

DNA Extraction Method:

DNeasy Mericon Food Kit

(Qiagen, Hilden,

Germany)

Primer:

•

Universal

primer used

were

researcher-

designed.

•

The primers

used for the pig

species were

researcher-

designed

Gene Targets:

•

16s rRNA

(Universal).

•

Cyt-

b (porcine

specific)

1 pg/µL

(22)

Gelatin

Capsule

Shell

Detection Method:

Duplex PCR

DNA Extraction Method:

DNeasy Mericon Food Kit

(Qiagen, Germany)

Primer:

Porcine

-specific

primers used were

researchers.

Gene Targets:

Cyt

b gene (Porcine

specific)

0.1% DNA or

0.1 gram/100

ml DNA

(10)

Health

Supplement

Gelatin

Capsule

Shell

Detection Method:

PCR with WGA (Whole

Genome Amplification)

DNA Extraction Method:

DNeasy® Blood & Tissue

or DNeasy® Plant kit

(Qiagen)

Primer:

SFI11

-Pig-F and

SFI11

-Pig-R

Gene Targets:

16s rRNA gene

(Porcine specific)

0.01ng/µL

(9)

395

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Gelatin

Capsule

Shell

Detection Method:

PCR RFLP

DNA Extraction Method:

Genomic DNA Mini Kit

(Geneaid, Taiwan)

Primer:

Porcine

-specific

primers used were

researchers.

Gene Targets:

Cyt

-b1 and Cyt-b2

genes (porcine

specific)

Restriction

Enzymes:

BsaJ

3000 µg/mL

(23)

Table 3. Advantage and Disadvantage of the Method from Scientific Journal Data

Method

Analysis Advantage Disadvantage

PCR

MyTaq

DNA

Polymerase

(16)

MyTaq DNA

Polymerase

enables accurate and

sensitive

detection of porcine DNA

collagen cream

cosmetic

products through efficient

and

specific DNA amplification

higher temperatures.

The heat resistance

the enzyme can

result

in non-

specific

amplification that

may

affect the

method's

specificity.

Real-time

PCR

(7,12,17,19)

A specific and

sensitive

method for detecting

porcine

materials in gelatin,

gelatin

capsules, and cosmetics

with

quantitative and real-

time

monitoring capabilities.

Complex

modifications

are

necessary to

enhance

the

interaction

between gelatin

and

DNA

during

extraction,

and

selecting the

right

method is crucial

for

accurate detection.

396

R. I. P. Syafitri et al / Indo J Pharm 5 (2023) 385-405

PCR-

RFLP

(20,23)

It can detect

products

containing pork, beef, and

fish

in one test without any cross

reaction between the

main

targets (cow, pork and

fish

DNA).

It requires complexity

primer design and

high

technical expertise

for

interpreting

restriction

pattern, as this can affect

the

specificity and success of

the

amplification.

Duplex

PCR

(10,18)

It enables

simultaneous

detection of multiple

target

DNA sequences in a

single

reaction, thus saving time

and

improving efficiency.

There is a risk of

false

positives due to non-

specific

amplification caused

cross-reactivity or

sample

contamination.

TaqMan

Qualitat

ive

PCR

Multiple

Probe

(21)

Specific, sensitive,

and

potentially cost-effective

test

for detecting 3 species at

once

(cow, pig and fish).

It is difficult to apply

for

samples

containing

genetically closely

DNA.

Convent

ional

PCR

(15)

Sensitive enough for

detecting

low percentage of bovine

and

porcine gelatin

It cannot be applied

for

quantitative analysis.

PCR

with

Loop-

Mediate

Isother

mal

Amplifi

cation

(LAMP)

(22)

LAMP is a cost-effective

and

accessible method for

porcine

detection as it does not

require

thermal cycling like

PCR,

operating at a

constant

temperature.

LAMP requires

careful

primer design to

ensure

specificity and accuracy,

improper design can lead

non-specific

amplification

and false-positive results.

PCR

with

Whole

Genome

Amplifi

cation

(WGA)

(9)

WGA can increase

the

quantity of amplifiable

DNA,

thereby enhancing

the

sensitivity and accuracy

detection.

WGA in PCR can increase the risk

of cross

-contamination from other

positive samples.

4. Discussion

4.1 DNA Extraction/Isolation

Methods

DNA isolation in cosmetics,

particularly oil-based preparations, poses

challenges due to the manufacturing

process. Creams and similar cosmetics

undergo extensive processing, resulting in

minimal DNA content, making isolation

more difficult (24). In the study by Zabidi

et al. (16), the Genomic Wizard DNA

purification kit yielded DNA

concentrations ranging from 3.252-4.222

ng/μL, with DNA purity ranging from

1.0-1.4 (optimal DNA purity value: 1.8-

2.0). The DNA concentration was

relatively low compared to DNA isolated

from processed food products (positive

control), and the low DNA purity could

be attributed to contamination with other

proteins. Cream-based cosmetic products

undergo extensive processing and contain

additives, resulting in a small amount of

DNA (24).

From several studies, it is known

that the most suitable kit for extracting

porcine DNA from cosmetic products is

the Power PrepTM DNA compared to

other method (7). The Power PrepTM

DNA extraction kit, which incorporates

chloroform, is considered more efficient

compared to other extraction kits.

Chloroform or hexane can be added to

fat-rich cosmetics like creams to alleviate

PCR process inhibitions, enhancing

detection capabilities (25–27).

The DNeasy Mericon Food Kit is

the most used commercial kit for isolating

pig DNA from gelatin samples or gelatin

capsule shells, as indicated in Table 2.

Additionally, some studies have

developed specific DNA extraction

methods to increase the yield of DNA for

analysis. Yang et al. (11) compared DNA

quality and PCR success rates of four

commercial kits used for DNA extraction.

The QuickGene DNA Tissue Kit L is the

top-performing commercial kit for

extracting porcine DNA from gelatin. It

demonstrates the highest DNA

concentration, optimal A260/280 value

indicating purity, superior amplification

efficiency, and efficient handling of

gelatin samples in terms of speed (11).

In addition, Mohamad et al. (12)

optimized the DNA extraction from

gelatin and gelatin capsules by modifying

the DNA-gelatin interaction. They used

the MasterPure DNA Purification Kit and

found that adjusting the pH to 8.5

significantly increased the amount of

extracted DNA compared to samples

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4.2 Porcine DNA Detection Method

Polymerase Chain Reaction is an

in vitro technique that amplifies target

DNA fragments. It involves denaturation,

annealing, and elongation stages using

polymerase enzymes and

deoxynucleotide triphosphates (dNTPs).

Specially designed primers determine the

target DNA fragment (14).

Identification of halal products

based on mitochondrial DNA (mt-DNA)

is estimated to be more accurate because

it is more specific, reproducible, sensitive

primers, affordable and has a faster

processing time (28). In addition, the use

of mt-DNA is also due to the high amount

of mt-DNA found in mitochondria

compared to DNA in nuclear cells or

nuclear DNA (7). On the other hand,

identification based on nuclear

(chromosomal) DNA as the target gene is

claimed to be less stable and subject to

changes in the number of copies during

the evolutionary process (29,30), but the

use of nuclear DNA has the advantage of

helping the PCR amplification process

from degraded DNA extraction due to

lengthy processing of samples (31,32).

Mohamad et al. (19) conducted a study

comparing the detection of porcine DNA

in soft capsule samples using

mitochondrial DNA and nuclear DNA

gene targets. The findings showed that

nuclear DNA detection provided a more

sensitive limit of detection (1 pg gelatin

DNA) compared to mitochondrial DNA

(10 pg gelatin DNA).

In the PCR method, optimization

is necessary at each stage, particularly in

determining the annealing temperature

and time. Annealing refers to the binding

of primers to DNA fragments. Khayyira

et al.’s (18) found that the optimal

annealing temperature for duplex PCR

with porcine DNA specific primers was

56°C. Temperatures of 55°C and 57°C

resulted in non-specific bands, possibly

due to primer non-specificity at low

temperatures and denaturation at high

temperatures.

Various PCR methods and their

modifications, such as Real-Time PCR,

PCR-RFLP, Multiplex-PCR, Duplex-

PCR, and MyTaq DNA Polymerase PCR,

have been utilized in the detection of

porcine DNA in cosmetic products and

health supplements. Comparison of

various methods can be seen in Table 3,

including their advantages and

disadvantages. These modifications aim

to enhance the detection sensitivity and

accuracy of porcine DNA.

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Real-Time PCR

Real-Time PCR is a technique

used to quantify target DNA. RT-PCR or

qPCR amplifies as well as calculates the

number of target DNA molecules

amplified. The principle of qPCR is the

use of fluorescent dyes as probes or label

dyes (14). Kim et al. (7) used RT-PCR to

quantify pig DNA and obtained a

threshold cycle (Ct) value. However, a

high Ct value can potentially be a false

positive due to amplification artifacts or

residual fluorescence. To validate the Ct

value, agarose gel analysis is

recommended to confirm the presence of

the correct target gene.

Multiplex and Duplex PCR

Multiplex and Duplex PCR are

modified amplification methods that

allow for simultaneous detection of

multiple loci within a single PCR

reaction. Multiplex PCR utilizes several

specific primers (more than 2), while

Duplex PCR utilizes 2 specific primers to

amplify different target sequences (14).

Khayyira et al. (18) carried out the

detection of pig DNA using the Duplex

PCR method. This study proves that the

method can provide sensitivity and

specificity in the detection of porcine

DNA at low concentrations. In addition to

its use for the analysis of halal products,

the use of Multiplex and Duplex PCR has

been applied in many areas of DNA

testing such as gene deletion analysis,

mutation and polymorphism detection,

quantitative analysis, identification of

viruses and parasitic bacteria (28,33,34).

The advantage of using this method is

that it can test 3 DNA contents at once in

one test (20).

PCR-RFLP

PCR-RFLP (Polymerase Chain

Reaction-Restriction Fragment Length

Polymorphism) involves treating PCR

amplicons with specific restriction

enzymes to generate DNA fragments of

varying sizes. These fragments are then

separated by gel electrophoresis. The

method is known for its simplicity,

making it accessible to those with limited

experience in molecular biology.

However, PCR-RFLP requires specific

restriction enzymes and faces challenges

in accurately identifying variations when

multiple SNPs (Single Nucleotide

Polymorphisms) are targeted

simultaneously (35). In the research

journals used as data in this review, there

are 3 studies using PCR-RFLP to detect

porcine DNA in samples. Of the 3 studies,

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R. I. P. Syafitri et al / Indo J Pharm 5 (2023) 385-405

2 studies used the restriction enzyme

BsaJI and 1 study used the restriction

enzyme BsaAI.

Modified Probe Real-Time PCR

Several studies have utilized

commercial PCR kits, including MyTaq

DNA Polymerase PCR, TaqMan Probe

real-time PCR, and TaqMan Probe

Multiplex Qualitative PCR. These kits

employ similar PCR principles with the

addition of DNA probes that specifically

bind to DNA targets, exhibiting strong

affinity (36). The type of probe used is

the TaqMan probe, which is an

oligonucleotide sequence which at the 5'

end has a fluorescent label dye and the 3'

end has a quencher label dye (37). The

advantage of this TaqMan probe is that it

can be easily synthesized and designed,

but when the probe is not designed

optimally, secondary chains such as

hairpins, dimers, runs, and repeats can be

formed during the PCR process (38).

Modified Amplification PCR

In addition to PCR methods,

other amplification techniques such as

Loop Mediated Isothermal Amplification

(LAMP) and Whole Genome

Amplification Kit (WGA) have been

utilized to increase the amount of

extracted DNA for detection purposes.

These methods offer alternative

approaches for DNA amplification in

various studies. Tasrip et al. (22) utilized

LAMP for DNA amplification. The

LAMP method involves thermal

amplification of DNA using specific

primers, resulting in increased

specificity of detection due to the

recognition of multiple target DNA

regions by four primers. Meanwhile, Lee

et al. (9) used the WGA Kit to amplify

the extracted DNA. WGA offers

unbiased amplification of the entire

genome, high yield, and scalability. It is

suitable for various applications such as

RFLP analysis, cloning, and DNA

sequencing, and can amplify small

starting material amounts (39–41). In the

analysis conducted by Lee et al. (9), it

was found that from samples treated

with WGA, DNA fragments were

produced in the range of 100 to 1000 bp.

5. Conclusion

Based on the collected research,

there are many cosmetic and health

supplement products that contain porcine

DNA. Therefore, testing for porcine DNA

is crucial to ensure the halal quality of the

products. DNA-based methods, including

the Polymerase Chain Reaction (PCR)

400

R. I. P. Syafitri et al / Indo J Pharm 5 (2023) 385-405

and its modifications, has been

proven to detect the presence of porcine DNA

in products like cosmetics and health

supplements. The selection of DNA detection

and isolation methods depends on the type of

sample under examination, thus enhancing the

accuracy and success of porcine DNA

detection.

401

R. I. P. Syafitri et al / Indo J Pharm 5 (2023) 385-405

References

1. Hanim Yusuf A, Abdul Shukor S,

Salwa Ahmad Bustamam U. Halal

Certification vs Business Growth of Food

Industry in Malaysia. JOEBM.

2016;4(3):247–51.

2. Maslul S, Utami IR. Halal Food

Products Labeling According to Islamic

Business Ethics and Consumers

Protection Law. Al-Iktisab [Internet].

2018 Nov 1 [cited 2023 Jun 4];2(2).

Available from:

https://ejournal.unida.gontor.ac.id/index.p

hp/aliktisab/article/view/3900

3. Hermanto S, Sumarlin LO,

Fatimah W. Differentiation of Bovine and

Porcine Gelatin Based on Spectroscopic

and Electrophoretic Analysis. JFPS.

2013;1(3).

4. Mukherjee S. Global Halal: Meat,

Money, and Religion. Religions. 2014 Jan

29;5(1):22–75.

5. Marianela CR, Daniel AA. Gelatin

and non-gelatin soft gel capsules: A

review. 2021;

6. Hassan N, Ahmad T, Zain NM,

Awang SR. Identification of bovine,

porcine and fish gelatin signatures using

chemometrics fuzzy graph method. Sci

Rep. 2021 May 7;11(1):9793.

7. Kim YS, Yu HK, Lee BZ, Hong

KW. Effect of DNA extraction methods

on the detection of porcine ingredients in

halal cosmetics using real-time PCR.

Appl Biol Chem. 2018 Oct;61(5):549–55.

8. Waskitho D, Lukitaningsih E,

Sudjadi, Rohman A. Analysis of Lard in

Lipstick Formulation Using FTIR

Spectroscopy and Multivariate

Calibration: A Comparison of Three

Extraction Methods. J Oleo Sci.

2016;65(10):815–24.

9. Lee JH, Kim MR, Jo CH, Jung

YK, Kwon K, Kang TS. Specific PCR

assays to determine bovine, porcine, fish

and plant origin of gelatin capsules of

dietary supplements. Food Chemistry.

2016 Nov;211:253–9.

10. Nikzad J, Shahhosseini S,

Tabarzad M, Nafissi-Varcheh N, Torshabi

M. Simultaneous detection of bovine and

porcine DNA in pharmaceutical gelatin

capsules by duplex PCR assay for Halal

authentication. DARU J Pharm Sci. 2017

Dec;25(1):3.

11. Yang Y, Li L, Wang H, Liu M, Wu

Y. Development and verification of a

quantitative real‐time PCR method to

identify and quantify gelatin derived from

animal hide. Journal of Food Science.

2020 Sep;85(9):2762–72.

12. Mohamad NA, Mustafa S, El

Sheikha AF, Khairil Mokhtar NF, Ismail

A, Ali ME. Modification of gelatin-DNA

interaction for optimised DNA extraction

from gelatin and gelatin capsule:

Optimised DNA extraction from gelatin. J

Sci Food Agric. 2016 May;96(7):2344–

51.

13. Nooratiny I, Sahilah AM, Alfie

ARA, Farouk MYMohd. DNA extraction

from ghee and beef species identification

using polymerase chain reaction (PCR)

assay. IFRJ. 2013;20(5):2959–61.

14. Hidayati H, Hasbullah H, Hasri S.

Methods for Detection of Foods,

Cosmetics, and Drugs Through A

Mitochondrial DNA Analysis (An

Overview of the Molecular and the

Qur’anic Aspects). In: Proceedings of the

Proceedings of the 19th Annual

International Conference on Islamic

Studies, AICIS 2019, 1-4 October 2019,

Jakarta, Indonesia [Internet]. Jakarta,

Indonesia: EAI; 2020 [cited 2023 Apr

29]. Available from:

http://eudl.eu/doi/10.4108/eai.1-10-

2019.2291746

15. Shabani H, Mehdizadeh M,

Mousavi SM, Dezfouli EA, Solgi T,

Khodaverdi M, et al. Halal authenticity of

gelatin using species-specific PCR. Food

Chemistry. 2015 Oct;184:203–6.

16. Zabidi AR, Fauzi FN, Abd Razak

FN, Rosli D, Jamil MZM, Wan Ibrahim

WK, et al. Screening porcine DNA in

collagen cream cosmetic products. Food

Res. 2020 Feb 10;4(S1):151–6.

17. S Abd-Gani S, Mustafa S, N

Mohd Desa M, F Khairil Mokhtar N, K

Hanapi U, Zakaria Z, et al. Detection of

Porcine Adulteration in Cosmetic Cream

Formulation via TaqMan Probe Real-

Time Polymerase Chain Reaction. IJET.

2019 Dec 24;7(4.14):112.

18. Khayyira AS, Estepane VM,

Malik A. RAPID PCR–BASED

DETECTION OPTIMIZATION OF

PORCINE DNA IN GELATIN

CAPSULE SHELL. Int J App Pharm.

2018 Nov 22;10(6):217.

19. Mohamad NA, Mustafa S, Khairil

Mokhtar NF, El Sheikha AF. Molecular

beacon-based real-time PCR method for

detection of porcine DNA in gelatin and

gelatin capsules: Can a molecular beacon

approach be used to authenticate the

origin of gelatin? J Sci Food Agric. 2018

Sep;98(12):4570–7.

20. Sultana S, Hossain MAM,

Naquiah NNA, Ali MdE. Novel multiplex

PCR-RFLP assay discriminates bovine,

porcine and fish gelatin substitution in

Asian pharmaceuticals capsule shells.

Food Additives & Contaminants: Part A.

2018 Sep 2;35(9):1662–73.

402

R. I. P. Syafitri et al / Indo J Pharm 5 (2023) 385-405

21. Sultana S, Hossain MAM, Azlan

A, Johan MR, Chowdhury ZZ, Ali MdE.

TaqMan probe based multiplex

quantitative PCR assay for determination

of bovine, porcine and fish DNA in

gelatin admixture, food products and

dietary supplements. Food Chemistry.

2020 Sep;325:126756.

22. Tasrip NA, Mohd Desa MN,

Khairil Mokhtar NF, Sajali N, Mohd

Hashim A, Ali MdE, et al. Rapid porcine

detection in gelatin-based highly

processed products using loop mediated

isothermal amplification. J Food Sci

Technol. 2021 Dec;58(12):4504–13.

23. Malik A, Sutantyo ML, Hapsari I,

Sinurat AV, Purwati EM, Jufri M, et al.

Simultaneous identification and

verification of gelatin type in capsule

shells by electrophoresis and polymerase

chain reaction. Journal of Pharmaceutical

Investigation. 2016 Aug;46(5):475–85.

24. Yang H, Shu Z. The extraction of

collagen protein from pigskin. 2014;

25. Terry CF, Harris N, Parkes HC.

Detection of genetically modified crops

and their derivatives: critical steps in

sample preparation and extraction. J

AOAC Int. 2002;85(3):768–74.

26. Hubbard WD, Sheppard AJ,

Newkirk DR, Prosser AR, Osgood T.

Comparison of various methods for the

extraction of total lipids, fatty acids,

cholesterol, and other sterols from food

products. J Amer Oil Chem Soc. 1977

Feb;54(2):81–3.

27. Hu Q, Liu Y, Yi S, Huang D. A

comparison of four methods for PCR

inhibitor removal. Forensic Science

International: Genetics. 2015 May;16:94–

28. Irine I, Nuraini H, Sumantri C.

Species Authentication of Dog, Cat, and

Tiger Using Cytochrome β Gene. Med

Pet. 2013 Dec;36(3):171–8.

29. Lovatt A. Applications of

quantitative PCR in the biosafety and

genetic stability assessment of

biotechnology products. Reviews in

Molecular Biotechnology. 2002

Jan;82(3):279–300.

30. Mishra S, Whetstine JR. Different

Facets of Copy Number Changes:

Permanent, Transient, and Adaptive.

Molecular and Cellular Biology. 2016

Apr 1;36(7):1050–63.

403

R. I. P. Syafitri et al / Indo J Pharm 5 (2023) 385-405

31. Walker JA, Hughes DA, Hedges

DJ, Anders BA, Laborde ME, Shewale J,

et al. Quantitative PCR for DNA

identification based on genome-specific

interspersed repetitive elements.

Genomics. 2004 Mar;83(3):518–27.

32. Furda A, Santos JH, Meyer JN,

Van Houten B. Quantitative PCR-Based

Measurement of Nuclear and

Mitochondrial DNA Damage and Repair

in Mammalian Cells. In: Keohavong P,

Grant SG, editors. Molecular Toxicology

Protocols [Internet]. Totowa, NJ: Humana

Press; 2014 [cited 2023 Jun 4]. p. 419–37.

(Methods in Molecular Biology; vol.

1105). Available from:

http://link.springer.com/10.1007/978-1-

62703-739-6_31

33. Henegariu O, Heerema NA,

Dlouhy SR, Vance GH, Vogt PH.

Multiplex PCR: Critical Parameters and

Step-by-Step Protocol. BioTechniques.

1997 Sep;23(3):504–11.

34. Markoulatos P, Siafakas N,

Moncany M. Multiplex polymerase chain

reaction: A practical approach. J Clin Lab

Anal. 2002;16(1):47–51.

35. Hashim Ali K, Mohsin Ansari M,

Ali Shah F, Ud Din F, Abdul Basit M,

Kim JK, et al. Enhanced dissolution of

valsartan-vanillin binary co-amorphous

system loaded in mesoporous silica

particles. Journal of Microencapsulation.

2019 Jan 2;36(1):10–20.

36. Yowani SC, Jennifer T, Ayu NCY,

Putu S. Desain Pelacar DNA In Silico

sebagai Pendeteksi Resistensi

Fluroroquinolone pada Isolat Multi Drug

Resistant Tuberculosis. Cakra Kimia

(Indonesian E-Journal of Applied

Chemistry). 2018;7(2).

37. Rahmaryani IGAAS, Ariani NK,

Dewi DSW, Ani NKS, Sundari AA,

Hartati KWY, et al. DNA Probe Design

for Detection Mutation at Codon 315 In

katG Gene of Mycobacterium

Tuberculosis to Real-Time Polymerase

Chain Reaction. Journal of Health

Sciences and Medicin. 2017 Sep;1(2).

38. Navarro E, Serrano-Heras G,

Castaño MJ, Solera J. Real-time PCR

detection chemistry. Clinica Chimica

Acta. 2015 Jan;439:231–50.

39. Lasken RS, Egholm M. Whole

genome amplification: abundant supplies

of DNA from precious samples or clinical

specimens. Trends in Biotechnology.

2003 Dec;21(12):531–5.

404

R. I. P. Syafitri et al / Indo J Pharm 5 (2023) 385-405

40. Wang X, Liu Y, Liu H, Pan W,

Ren J, Zheng X, et al. Recent advances

and application of whole genome

amplification in molecular diagnosis and

medicine. MedComm [Internet]. 2022

Mar [cited 2023 Jun 4];3(1). Available

from:

https://onlinelibrary.wiley.com/doi/10.100

2/mco2.116

41. Jäger R. New Perspectives for

Whole Genome Amplification in Forensic

STR Analysis. IJMS. 2022 Jun

25;23(13):7090.

405

R. I. P. Syafitri et al / Indo J Pharm 5 (2023) 385-405

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