Study of the Modification of Loop-mediated Isothermal Amplification

(LAMP) using Taq Polymerase for Halal Testing

Rosy Hutami

, Mira Suprayatmi

, Nida Idzni

, Raafqi Ranasasmita

, Henny Nuraini

and Joko Hermanianto

Department of Food Technology and Nutrition, Djuanda University, Bogor, Indonesia

Halal Laboratory, The Assessment Institute for Foods, Drugs And Cosmetics, Bogor, Indonesia

Department of Animal Production and Technology, Bogor Agricultural University Bogor, Indonesia

Department of Food Science and Technology, Bogor Agricultural University Bogor, Indonesia

Keywords: LAMP, Taq Polymerase, Enzyme, Bst, Betaine, Halal.

Abstract: Loop-Mediated Isothermal Amplification (LAMP) is a new method in nucleic acid analysis that amplifies

target in isothermal or constant conditions. This method is suitable for analysis with low-resource condition.

Commonly, this method needs a polymerase enzyme that has strand displacement activity such as Bacillus

stearothermopilus (Bst) polymerase. But, this enzyme was rarely available in the laboratory. Taq polymerase

is an polymerase enzyme that usualy available in molecular laboratory, easier to be accessed, and has a lower

cost comparing to the Bst polymerase. This research conducted with Taq polymerase in order to study the

compatibility of taq polymerase in amplificating the deoxyribonucleic acid (DNA) with LAMP method. We

used cytochrome b (cyt b) from pork as terget gene for halal detection need. For conducting the strand

displacement activity in DNA amplification, we used denaturation process (95

C), following by annealing

(65

C), and enzyme inactivation (80

C). The result showed there was no band appeared on the agarose after

electrophoresis. It was suggested that the Taq polymerase was not suitable for LAMP analysis although it has

been combined with denaturation process. The LAMP should be conducted with a suitable polymerase that

has strand displacement activity and some supporting reagents such as betaine and LAMP buffer that can

strengthen the reaction under isothermal condition.

1 INTRODUCTION

Halal food is currently one of the centers of attention

in the global food industries. Muslims are obliged to

consume only halal and thayyib foods and drinks. In

Islamic perspective, basically everything on earth is

allowed to be consumed except those that are

prohibited, and among those that are forbidden are

pigs and their derivatives (Al-Baqarah: 168, Al-

Baqarah: 173, Al An'am: 145, Al-Maidah: 3, An

Nahl: 115).

Today, various technologies for testing the

authenticity of a product are progressing rapidly.

Many analytical methods are developed and offer fast

and authentic results, one of which is a DNA-based

method. Methods of analysis using DNA have several

advantages, including DNA can be found in all cell

types in each individual with identical genetic

information, DNA is a stable molecule in the

extraction process, and DNA analysis is very likely to

be done from several different types of samples (Jain,

2004).

One of the most widely used nucleic acid

amplification technologies (DNA and RNA) is

Polymerase Chain Reaction (PCR) technology. PCR

is one of the most widely used methods of DNA

propagation in authentication and can also copy

nucleic acids millions of times from the initial

reaction. However, conventional PCR has the

disadvantage that this method requires the separation

of post-PCR products using electrophoresis gel which

is time-consuming and only semi-quantitative

(Kumar, 2007). These weaknesses can be overcome

by real-time PCR using a fluorescence system so that

the amplification results can be seen directly. Real-

time PCR has a disadvantage that is the expensive

price so that not many laboratories in Indonesia have

it. Because the methods commonly used for DNA

detection have disadvantages, alternative methods

that can detect DNA with the same high sensitivity as

Real-time PCR are needed but use simple and real-

Hutami, R., Suprayatmi, M., Idzni, N., Ranasasmita, R., Nuraini, H. and Hermanianto, J.

Study of the Modiﬁcation of Loop-mediated Isothermal Ampliﬁcation (LAMP) using Taq Polymerase for Halal Testing.

DOI: 10.5220/0009940721912198

In Proceedings of the 1st International Conference on Recent Innovations (ICRI 2018), pages 2191-2198

ISBN: 978-989-758-458-9

2191

time equipment, so they can be applied in the field or

in laboratories with limited resources.

The study of identification of pork protein

contamination in food products such as meat can be

tested using several methods, namely the PCR

method, PDK (Porcine Detection Kit), and ELISA

(Enzyme-Linked Immunosorbent Assay). Ardi

(2012) has analyzed the presence of pig

contamination in meatball products sold in the market

by PCR method, and found a minimum level of pig

contamination that can be identified by PCR

technique of 0.5%. Rasyid (2015) has analyzed the

contamination of pork in beef products using real-

time PCR with the hydrolysis probe method, and

stated that by using real-time PCR and using the

hydrolysis probe method can amplify DNA from

meatballs using specific primers of cattle and pork in

mitochondrial cytochrome B area with 120 bp and

131 bp amplicons using annealing temperature in

61oC and 60oC.

In this study, the inspection method of a halal

product to be developed is the Loop-Mediated

Isothermal Amplification (LAMP) method. The

LAMP method is one of the molecular diagnostic

techniques that has been developed from 1999 in

Japan. The LAMP technique uses DNA amplification

at a fixed temperature, so that the use of expensive

thermocycler devices is not needed.

In the study using the LAMP method, Wilopo et

al. (2015) stated that the LAMP method can be used

as an alternative examination in detecting blaTEM

genes, especially in areas with limited laboratory

infrastructure. As for, Soleha (2015) states that the

LAMP method is very specific and has a high

sensitivity, fast, and economical. LAMP has a high

selectivity because it recognizes the target 6 different

sequences at the beginning of the reaction. Because

of the advantages of the LAMP method, it is

necessary to explore the method of detection of pork

DNA using the LAMP method by using the Taq

Polymerase enzyme, because the Taq Polymerase

enzyme is easily access, more economical prices, and

commonly used in DNA-based sample testing.

2 MATERIALS AND METHOD

The materials used in this study were pork, beef,

chicken, goat, fish, pork DNA primers for gene

fragments Cytochrome B, Sure Food DNA extraction

kits (R Biopharm AG, from buffer lysis, proteinase,

buffer bindings, pre wash buffer, wash buffer, elution

buffer), PCR GoTaq green master mix (Promega)

PCR reagent, DNA free aquades, agarose powder,

Tris-Borat-EDTA (TBE), FlouroSafe, loading dye

and DNA ladder 100bp.

The tools used in this research are analytical

balance (Precisa XB220A), Sorvall ST 16R (Thermo

Scientific) centrifuge, Genova Nano (Jenway)

spectrophotometer, thermoshaker heating block

Dipabis MHR 13, coolant, eppendorf 0.1-2.5μl

micropipette, micropipette eppendorf 0,5-10μl,

Eppendorf 100μl micropipette, Eppendorf 200μl

microphone, Eppendorf 1000μl micropipette, Gilson

20μl micropipette, vortex, PCR Gene Amp AB

system 9700, PCR (Esco), glassware, microwave,

magenetic stirrer, electrophoresis (Mupid- exu), UV-

Transilluminator (Alphalmager EP), plastic bags,

latex gloves, and spatula.

2.1 DNA Isolation

Isolation or DNA extraction was carried out using

extraction techniques with extraction kits issued by R

Biopharm AG (SureFood® PREP Basic Art kit. No.

S1052). Fresh meat to be extracted is pork, beef,

chicken, goat and fish. Then, the concentration and

purity of the DNA extract were analyzed using a

spectrophotometer and visualized by gel

electrophoresis.

Testing the quality of DNA extracts is done by

looking at the results of DNA visualization, DNA

concentration, and DNA purity resulting from

extraction. DNA visualization from extraction results

was carried out by electrophoresis on 1% gel. The gel

is made from 0.45 grams of agarose and 30 ml of

buffer solution (0.5 x TBE) is heated. The agarose

solution is left to cool a little while stirring with the

stirrer magnet, then adding 1.8 µl of Flouro Safe dye.

A total of 5 µl of DNA samples were dissolved in 1

µl of loading dye. Added 100 μl of 100 bp DNA

ladder as a tape measure on the gel. Electrophoresis

is carried out for 40 minutes at a constant voltage of

100 volts. After electrophoresis is complete, the gel is

taken to take photos using UV-Transilluminator.

Evaluating the quality of DNA extract was done

by testing the concentration and purity of DNA by

spectrophotometric analysis. The elution buffer

solution was used as blank as much as 2 µl of the

solution was dripped into a spectrophotometer, then a

sample of 2 µl of the solution was dripped into a

spectrophotometer and tested at a wavelength of 260

nm and 280 nm.

The obtained DNA extracts were diluted into

uniform DNA concentration. Dilution was aimed to

ensure that the amount of DNA to be amplified at the

next stage were uniform in each samples. The final

DNA concentration of all samples was diluted to 30

ng / µl and 50 ng / µl.

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2.2 Primer Design

Primer design was carried out with the NCBI program

to examine primer specificity, then specific DNA

segments were chosen as targets. The complete

sequence of this DNA segment was then downloaded.

Primer was made by using the Primer Explorer

version 5 program and produced 4 primer sets for

LAMP. Each primer set was checked for specificity

with BLAST alignment; examination of melting

temperature, self-complimentary and secondary

structure with Oligo Analyzer 3.1; and primer-dimer

checks between primers with the Thermo Multiple

Primary Analyzer.

2.3 DNA Amplification

This research stage is the stage of DNA amplification

by using 4 primers through denaturation (95 ° C),

annealing (65 ° C), and final extension (80 ° C)

stages. The purpose of this stage was to determine the

success of the LAMP method with modifications to 4

primers F3, B3, FIP, BIP with Cytochrome B DNA

template.

3. RESULT AND DISCUSSION

3.1 DNA Isolation

DNA concentrations ranged from 46.15 to 455.76 ng

/ μl. The amount of DNA produced was influenced by

several factors which are at the time of extraction and

also the condition of the sample. Komalasari (2009)

states that the concentration of DNA extraction was

influenced by two factors, namely the extraction

speed and the composition of buffer lysis adition.

The DNA extract concentration was not uniform.

Therefore, then dilution was conducted to uniform the

sample concentration. In this study, DNA extracts

were diluted up to of 30 ng / μl and 50 ng / μl

concentration. This concentration range was

commonly used in the PCR process. The use of non-

dense DNA concentrations can also avoid primer

attachment errors when the target DNA amplification

occurs.

DNA purity with absorbance ratio λ260 / λ280 has

a good result between 1.822 to 2.099. According to

Sambrook et al., (1989) and Muladno (2010) DNA

isolates can be said as pure and have met the

requirements required for molecular analysis when

the ratio λ260 / λ280 ranges between 1.8-2.0.

Extraction results with a ratio of 1.8 to 2.0 are high

purity DNA and are not contaminated with protein

residues. A value of 260 nm is the maximum value of

DNA that can absorb the light. This value can be used

to estimate the concentration of DNA. The value of

280 is the maximum value of protein residues that can

absorb light.

The extraction method used refers to the

extraction method issued by the Biopharm AG R

(SureFood® PREP Basic Art kit. No. S1052) by

making several modifications. This method of DNA

isolation or extraction used an extraction buffer that

was ready to use. In SureFood kit, the filtration

equipment used was spin filter, there were two types

of spin filters, namely clear spin filter and yellow spin

filter. Cells were lysed by using a lysis buffer. The

principle of lysis method was the destruction of cell

walls without having to damage the desired DNA.

Therefore, cell wall destruction was generally done

by breaking the cell wall using a lysis buffer

(Handoyo and Rudiretna, 2000). Then the cell

components, especially proteins, were destroyed by

using Proteinase-K (protease enzyme). Then, binding

the nucleic acids on spin filters using binding buffer,

filtered and bound of nucleic acids, and washed

nucleic acid using washing buffer (pre-wash buffer

and wash buffer). In the final stage the DNA was

dissolved in the elution buffer.

According to Sunarno et al., (2014), SureFood's

commercial extraction kit has the same principles as

other commercial extractions that use the principle of

mini column or DNA filtration. DNA extraction using

the mini column principle is the most common

extraction method because the results obtained are

very good with a process that is not too long when

compared to the phenol-chloroform method and a

very cheaper cost compared to the magnetic beads

method.

3.2 Primer Design

The primer design steps obtained several primer

pairs. Each primer candidates were tested for

specificity in pork DNA using the BLAST alignment

program from NCBI. The specificity test result by

BLAST alignment program can be seen in Table 1 to

Table 4.

Table 1. Primer Candidate 1

Primer Sequence Primer Specificity

F3 CCCTGAAT

CACCCGTA

Sus scrofa, Sus scrofa

breed, Sus

domesticus,

Prionailurus

bengalensis, Neofelis

nebulosa

mitochondrion, Lutra

lutra, Felis nigripes.

Study of the Modiﬁcation of Loop-mediated Isothermal Ampliﬁcation (LAMP) using Taq Polymerase for Halal Testing

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Primer Sequence Primer Specificity

B3 TGGTTTTT

GGTTATAC

TACTGC

Sus scrofa breed,

Podomys floridanus,

Sus Scrofa, Priodontes

maximus,

Chaetophractus

vellerosus, Sus

Barbatus,

F2 AAATTACT

CAATCCCC

AAGC

Sus scrofa breed, Sus

domesticus, Sus

Scrofa, Sus cebifrons.

F1C TATGCATT

GAAGGAA

GAGGAAG

TAG

Sus scrofa breed, Sus

Scrofa

B2 CATGGCTA

CTGAGATG

TACC

Sus scrofa breed, Sus

Scrofa, Diplodia

corticola, Sus

verrucosus.

B1c CAGAAAC

AAATGCTC

CAAAAAC

AGT

Sus scrofa breed, Sus

domesticus, Sus scrofa

breed, Sus Scrofa

Table 2. Primer Candidate 2 (Hutami et al. 2017)

Primer Sequence Primer

Specificity

F3 GTCTTATTAG

AAACTCAAAC

CTCA

Sus scrofa breed,

Sus domesticus,

Sus Scrofa

B3 TTTTCTTCTAA

ACCCTCTCCTA

Sus scrofa breed,

Sus domesticus,

Sus Scrofa

F2 GGGTACATCT

CAGTAGCCAT

Sus scrofa breed,

Sus domesticus,

Sus Scrofa

F1C TGGTGTTTTTG

ATTTATTTGGG

GGG

Sus scrofa breed,

Sus domesticus,

Sus Scrofa

B2 TGGACTTGGG

TTGATTGT

Sus scrofa breed,

Sus domesticus,

Sus Scrofa, Sus

cebifrons, Sus

verrucosus,

Tursiops

truncates.

B1c CCTAAAAAAG

ACCCACCAAA

ATTCA

Sus scrofa breed,

Sus domesticus,

Sus Scrofa,

Capra hircus,

ovis aries,

Tayassu pecari,

Pecari tajacu,

Catagonus

wagneri,

Bootherium

bombifrons,

Capra aegagrus,

Ovis ammon.

Table 3. Primer Candidate 3

Primer Sequence Primer Specificity

F3 TCAACTACAA

GAACCTTAAT

GAC

Sus scrofa breed,

Sus domesticus,

Sus Scrofa,

Delphinapterus

leucas, Ursus

arctos, Ursus

spelaeus, Ursus

ingressus,

Nannoperca

variegate.

B3 AGCTGTTGTT

GTGTCTGA

Sus scrofa breed,

Sus Scrofa,

Acanthochromis

polyacanthus,

Calomyscus

bailwardi,

Caenorhabditis

elegans.

F2 AACATCCGA

AAATCACACC

Sus scrofa breed,

Sus domesticus,

Sus scrofa, Lagopus

lagopus,

Myonycteris sp.,

Eonycteris spelaea

Sphaerias blanfordi,

Epomophorus

minimus, Epomops

buettikoferi.

F1C TGGGAGGTC

AATGAATGC

Sus scrofa breed,

Sus scrofa, Rusa

unicolor hainana,

Nanger dama

mhorr, Eudorcas

rufifrons, Gazella

dorcas, Hippotragus

leucophaeus,

Tragelaphus

strepsiceros, Ovis

vignei blanfordi,

Rupicapra

rupicapra, Lemmus

trimucronatus,

Capricornis sp.

B2 TGTGTAATGT

ATTGCTAAGA

ACA

Sus scrofa breed,

Sus scrofa,

Phlebotomus

perniciosus,

B1c AACTTCGGTT

CCCTCTTAGG

Sus scrofa breed,

Sus domesticus,

Sus scrofa,

Macrognathus

semiocellatus,

Phlebotomus

perniciosus.

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Table 4. Primer Candidate 4

Prime

Sequence Primer Specificity

F3 ATTCATTGAC

CTCCCAGC

Sus scrofa breed, Sus

domesticus, Sus scrofa,

Hubei orthoptera virus,

Microtus arvalis, Canis

lupus, Chodsigoa

hoffmanni, Canis

himalayensis, Rusa

unicolor hainana,

Nanger dama mhorr,

Eudorcas rufifrons,

Gazella dorcas,

Tragelaphus buxtoni,

Ovis vignei blanfordi,

Rupicapra rupicapra

B3 TGTAGGTAGC

GAATAACTCA

Sus scrofa breed, Sus

scrofa, Phacochoerus

africanus.

F2 CATCTCATCA

TGATGAAACT

TCG

Sus scrofa breed, Sus

scrofa breed, Sus

domesticus, Sus scrofa,

Microtus transcaspicus,

Microtus arvalis

mystacinus, Alophoixus

finschii, Bullimus

luzonicus, Habromys

ixtlani, Oecomys

catherinae, Blarinomys

breviceps, Tragelaphus

buxtoni,

F1C AGAACAGGC

CTGTTAGGAT

TTGC

Sus scrofa

B2 CGTAATTTAC

GTCTCGACAG

Sus scrofa breed, Sus

scrofa, Potos flavus,

Phyllotis definitus,

Planigale ingrami,

Blarinomys breviceps,

Halichoeres

maculipinna,

Acrossocheilus

beijiangensis,

Gerbilliscus

nigricaudus, Myotis

bechsteinii,

Neophocaena

phocaenoides sunameri,

Proechimys roberti,

Peromyscus furvus,

Aphyosemion georgiae,

Myodes glareolus,

Necromys lilloi,

Ichthyoelephas

longirostris, Cribroheros

robertsoni, Neoromicia

robertsi, Embiotoca

jacksoni, Ronquilus

Prime

Sequence Primer Specificity

jordani, Psammomys

obesus.

B1c ACATTACACA

TCAGACACAA

CAACA

Sus scrofa breed, Sus

scrofa, Grammomys sp.

Primer selection was based on the level of primer

specificity of pork DNA. Thus, Primer Candidate 2

was chosen with the consideration that, compared to

the other primer pairs, Primer Candidate 2 was more

specific for each primer with the DNA Sus scrofa

(Wild boar). Although primer B2 cross-reacts with

dolphins, it can be ignored because this organism was

not as common as food. Meanwhile, B1c primers

cross react with goats, sheep, Bootherium bombifrons

(variants of the extinct bull) and wild variations of

pigs (Tayassu pecari, Tajacu and Catagonus

wagneri). B1c primer cross reaction can also be

ignored because there were still 6 other primers which

prevent cross-linking with other species. The

characteristic of Primer Candidate 2 showed at Table

Table 5. Characteristic of Primer Candidate 2

When compared with the requirements of primer

design according to Viljoen et al., (2005) Primer

Candidate 2 have fairly good feasibility, including

primers having a nucleotide length of 18-28 bp, Tm

ranging from 55 ° C-72 ° C, hairpin and dimers would

not occur, primers (F3, B3, FIP, F2, F1C, B2, B1C)

were free from self-annealing, whereas in BIP

primers they had the possibility of self-annealing,

because they had ΔG value of -9.43 kcal / mol. But

there were some conditions that are not fulfilled,

including the composition of G and C not 50-60% and

at the primary end -3 'dominated by bases A and T.

However, the primer can still be used in research.

This was evidenced by the results of amplification of

pork DNA with conventional PCR methods using

selected primers that was success. Visualization of

Study of the Modiﬁcation of Loop-mediated Isothermal Ampliﬁcation (LAMP) using Taq Polymerase for Halal Testing

2195

pork DNA amplification results with selected primers

is shown in Figure 1 and Figure 2.

Figure 1: Visualization of Amplified DNA by PCR

(replication 1), 1: Non Template Control, 2 : Non Template

Control, 3 : Marker, 4 : Pork DNA 50 ng/µl, 5 : Pork DNA

30 ng/µl, 6 : Pork DNA 50 ng/µl, 7: Pork DNA 30 ng/µl.

Appeared bands showed that the primers and the

reagent including Taq polymerase enzyme were

suitable for DNA amplification using PCR protocol

(Fig. 1 and Fig 2). Then, the modified LAMP was

performed to study whether the primers and Taq

polymerase will also work in the LAMP protocol.

Figure 2: Visualization of Amplified DNA by PCR

(replication 2), 1: Non Template Control, 2: Non Template

Control, 3 : Marker, 4 : Pork DNA 50 ng/µl, 5 : Pork DNA

30 ng/µl, 6 : Pork DNA 50 ng/µl, 7: Pork DNA 30 ng/µl.

The LAMP procedure that used in this research

was modified by the procedure that developed by

Kanchanaphum et al. (2014). As modification, we

used denaturation process in 95

C. The result of

modified LAMP was showed in Figure 3 and Figure

Figure 3: Visualization of Amplified DNA by LAMP with

Modification (replication 1); 1: marker, 2 : Pork DNA 50

ng/µl, 3 : Pork DNA 30 ng/µl, 4 : Pork DNA 50 ng/µl, 5:

Pork DNA 30 ng/µl, 6 : Non Template Control, 7 : Non

Template Control.

Figure 4: Visualization of Amplified DNA by LAMP with

Modification (replication 2); 1 : marker, 2 : Pork DNA 50

ng/µl, 3 : Pork DNA 30 ng/µl, 4 : Pork DNA 50 ng/µl, 5:

Pork DNA 30 ng/µl, 6 : Non Template Control, 7 : Non

Template Control.

According to the results, there was no bands

appeared in DNA visualization after DNA

amplification by modified LAMP method (Fig. 3 and

Fig. 4).

This was allegedly caused by several factors, due

to differences in the used procedure compared with

The LAMP procedure used by Kanchanaphum et

al. (2014). The first factor was the type of polymerase

used. The function of polymerase functions is to

catalyze the formation of a phosphodiester bond

between OH at the end of the 3' carbon with the

phosphate group from the added dNTP (Notomi et al.,

205 b

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2000). In the Kanchanaphum’s procedure, the

polymerase used is Bst polymerase that has strand

displacement activity (Notomi et al., 2000), while in

this modified LAMP procedure, the polymerase used

(Taq polymerase) does not have any strand

displacement activity. Enzymes used in PCR (e.g.,

Taq DNA polymerase) possess high thermostability

and robust polymerase activity but do not exhibit a

strong strand displacement activity and are therefore

not suitable for isothermal amplification methods

such as LAMP (Konstantin et al. 2018). The second

factor was the temperature used. In the LAMP

procedure (Kanchanaphum et al. 2014), DNA

denaturation temperature was not used. In this

modified LAMP procedure, denaturation temperature

was available in order to displace the DNA stand.

However, the duration for denaturating was less than

common denaturation duration in PCR while using

Taq polymerase (5 minutes of pradenaturation

following by 2 seconds of denaturation). In this study,

denaturation was only carried out for 2 minutes. So

that the heating stage at 95

C (denaturation) was

considered unsuccessful in doing strand

displacement. The third factor was the used of

betaine. In the LAMP protocol (Kanchanaphum et al.

2014), betaine was used, when in the modified LAMP

protocol was not. Betaine has an important role in

strand displacement activity, because the presence of

betaine (N, N, N-trimethylglyine or L-proline) can

help destabilize the double helix structure in DNA

(Notomi et al. 2000). The fourth factor was the used

of LAMP buffer. In this LAMP modified protocol,

buffer was not used, while in the LAMP protocol

(Kanchanaphum et al. 2014), it was used. LAMP

reaction needs a certain pH, and LAMP buffer will

provide it (Yang et al. 2006). The summary of method

used in LAMP (Kanchanaphum et al. 2014) and

modified LAMP in this research was showed in Table

Table 6: The Summary of Method between LAMP

(Kanchanaphum et al. 2014) and Modified LAMP

Components

Method

LAMP

Method

(Kanchanaphu

m et al. 2014)

Modified LAMP

Primer

4 Primer :

F3, B3, FIP,

BIP

4 Primer :

F3, B3, FIP, BIP

Reagents

- Bst

polymerase

- MgSO

- Betaine

- dNTP

- Taq

polymerase

- MgCl

- dNTP

- Aquades

bebas DNA

- LAMP

Buffer

- Aquades bebas

DNA

Temperatur

e and Time

Annealing :

T: 65

C; t: 45

min

Enzyme

Inactivation :

T: 80

C; t: 5

min

Denaturation :

T: 95

C; t: 2 min

Annealing :

T: 65

C; t: 45 min

Enzyme

Inactivation :

T: 80

C; t: 5 min

4 CONCLUSIONS

Modification of the LAMP method was carried out by

changing the type of polymerase used, eliminating the

use of betaine and LAMP buffer and adding

denaturation temperature to the amplification

process. Based on the results, Taq polymerase was

not suitable for LAMP method because it does not

have the strand displacement activity of DNA

structure, although it was combined with denaturation

process (95

C, 2 min). The absent of betaine and

LAMP buffer were suspected giving impact in the

failure of reaction, because betaine plays role in

destabilizing the double helical structure of DNA and

LAMP buffer plays role in giving the suitable pH for

LAMP reaction. Thus, Taq polymerase and

modification of some reagents were not suitable to be

applied in LAMP method.

ACKNOWLEDGEMENTS

We would like to thanks for the support of Ministry

of Research Technology and Higher Education of

Indonesia for Gave the Grant of This Research in

accordance with the Research Contract No.

1598/K4/KM/2017. We would like to thanks also for

Department of Animal Production and Technology,

Faculty of Animal Science, Bogor Agricultural

University, Indonesia and The Assesment Institute for

Foods, Drugs and Cosmetics, The Indonesian Council

of Ulama, Indonesia.

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