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 BANGLADESH RESEARCH PUBLICATIONS JOURNAL
ISSN: 1998-2003, Volume: 5, Issue: 4, Page: 425-439, July -August, 2011
Review Paper

LOOP MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) - AN

ALTERNATIVE TO POLYMERASE CHAIN REACTION (PCR)
Md. Fakruddin* 1

Md. Fakruddin (2011). Loop mediated Isothermal Amplification (LAMP) - An Alternative to

Polymerase Chain Reaction (PCR). Bangladesh Res. Pub. J. 5(4): 425-439. Retrieve from
http://www.bdresearchpublications.com/admin/journal/upload/09235/09235.pdf

Abstract
Loop-mediated isothermal amplification (LAMP) is a simple, rapid,
specific and cost-effective nucleic acid amplification method when
compared to PCR, nucleic acid sequence-based amplification, self-
sustained sequence replication and strand displacement amplification.
This protocol details an improved simple visual detection system for the
results of the LAMP reaction. This method employs a DNA polymerase
and a set of four specially designed primers that recognize a total of six
distinct sequences on the target DNA. Expensive equipments are not
necessary to acquire a high level of precision, and there are fewer
preparation steps compared to conventional PCR and real-time PCR
assays. In LAMP, a large amount of DNA is synthesized, yielding a large
pyrophosphate ion by-product. The reaction solution allows a
visualization of substantial alteration of the fluorescence during the one-
step amplification reaction, which takes 30–60 min. As the signal
recognition is highly sensitive, this system enables visual discrimination of
results without costly specialized equipment. This detection method
should be helpful in basic research on medicine and pharmacy,
environmental hygiene, point-of-care testing and more. It is an
established nucleic acid amplification method offering rapid, accurate,
and cost-effective diagnosis of infectious diseases. Considering the
advantages of rapid amplification, simple operation and easy
detection, LAMP has potential applications for clinical diagnosis as well
as surveillance of infectious diseases in developing countries without
requiring sophisticated equipment or skilled personnel.
Key Words: Nucleic acid amplification; Loop-mediated isothermal amplification
(LAMP); PCR; Application.
Introduction
Nucleic acid amplification is a valuable molecular tool not only in basic
research but also in application oriented fields, such as clinical medicine
development, infectious diseases diagnosis, gene cloning and industrial quality
control etc. (Xue-en Fang et al., 2008). Several amplification methods have been
developed already, such as polymerase chain reaction (PCR) (Saiki et al., 1985),
self-sustained sequence replication (3SR) (Guatelli et al., 1990), nucleic acid
sequence based amplification (NASBA) (Compton, 1991), strand displacement
amplification (SDA) (Walker et al., 1992) and rolling circle amplification (RCA)
(Lizardi et al., 1998) etc. Most of these methods can amplify targets by many
magnitudes of order using its own particular mechanism to re-initiate new rounds

Corresponding Author’s email: fakruddinmurad@gmail.com

Scientific Officer, Institute of Food Science and Technology (IFST), Bangladesh Council of Scientific
and Industrial Research (BCSIR), Dhaka.
Fakruddin 426

of DNA synthesis, but still have many shortcomings, including the requirement of
precision instruments and elaborate methods for product detection. LAMP is a
novel nucleic acid amplification method developed by Notomi et al, (2000)
which amplifies DNA with high specificity, sensitivity and rapidity under isothermal
condition using a set of four specially designed primers and a Bst DNA
polymerase. In this review article, we overview the current status of LAMP and
recent developments & future prospects associated with the method.
Rationale for the development of LAMP
PCR is the most widely used nucleic acid amplification method. But, it has
several limitations such as: 1.The usefulness of PCR methods can be limited by the
presence of PCR inhibitors in the analysis of real biological samples (including
food samples). The wide range of inhibitors (including organic and inorganic
substances such as detergents, antibiotics, phenolic compounds, enzymes,
polysaccharides, fats, proteins and salts) reduced or inhibited the amplification
efficiency (Wilson 1997; Al-Soud and Radstrom 1998) 2. PCR is labor-intensive and
often requires extensive sample preparation to eliminate amplification inhibitors.
3. PCR methods are also sensitive to contamination (Corless et al., 2000) 4. High-
cost, sophisticated instruments for amplification and detection of the amplified
products are required 5. Minimal 1 h is needed for the procedure. (Hung-Yueh et
al., 2005) 6. The other main limitation to PCR is the length of the region that can
be amplified. PCR works well over short stretches of DNA up to about 2 kb. 7. One of the
most fundamental limitations of PCR is that one need to have some DNA sequence
information before one begin. To overcome these limitations, development of a new
technique becomes urgent.
Principle of LAMP
LAMP relies on auto-cycling strand displacement DNA synthesis which is
carried out at 60- 65 °C for 45-60 min in the presence of Bst DNA polymerase,
dNTPs, specific primers and the target DNA template. The LAMP method employs
a DNA polymerase with high strand displacement activity and a set of four
specially constructed Primers (two inner and two outer primer) that recognize six
distinct sequences on the target DNA. The inner primers are called the forward
inner primer (FIP) and the backward inner primer (BIP) and each contains two
distinct sequences corresponding to the sense and antisense sequences of the
target DNA. One of the inner primer initiates LAMP reaction and the other is used
for self-priming in later stages. The mechanism of the LAMP amplification reaction
includes three steps: production of starting material, cycling amplification and
elongation, and recycling (Notomi et al. 2000).
After initiation by an inner primer, a pair of ‘outer’ primers then displaces
the amplified strand with the help of Bst DNA polymerase which has a high
displacement activity, to release a single stranded DNA, which then forms a
hairpin to initiate the starting loop for cyclic amplification. This serves as template
for DNA synthesis primed by the second inner and outer primers that hybridize to
the other ends of the target, which produces a stem-loop DNA structure. In
subsequent LAMP cycling one inner primer hybridizes to the loop on the product
and initiates displacement DNA synthesis, yielding the original stem-loop DNA and
a new stem-loop DNA with a stem twice as long. Amplification proceeds in
cyclical order, each strand being displaced during elongation with the addition
of new loops with every cycle. The cycling reaction continues with accumulation
of 109 copies of target in less than an hour. The final products are stem-loop DNAs
with several inverted repeats of the target and cauliflower-like structures with
multiple loops formed by annealing between alternately inverted repeats of the

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LAMP - An Alternative to PCR 427

target in the same strand (Notomi et al., 2000). The reaction can be accelerated
by using two extra loop primers (Nagamine et al. 2002).
The use of primers (recognition of six distinct sequence) in the initial steps of
LAMP and two primers (recognition of four distinct sequences) during the
subsequent steps ensures high specificity for target amplification. Moreover, in
LAMP four primers (six distinct sequences recognition) are simultaneously used to
initiate DNA synthesis from the original unamplified DNA to generate a stem-loop
DNA for subsequent LAMP cycling, during which the target is recognized by four
sequences. Therefore, target selectivity is expected to be higher than those
obtained in PCR (Notomi et al., 2000).
Reactions of LAMP:
For ease of explanation, the sequences (typically 23-24 nucleotides) inside
both ends of the target region for amplification in a DNA are designated F2c and
B2, respectively (Fig 1). Two inner sequences (typically 23-24 nucleotides) 40
nucleotide from the ends of F2c and B2 are designated F1c and B1 and two
sequences (17-21 nucleotide) outside the ends of F2c and B2 are designated as
F3c and B3. Forward inner primer (FIP) contains F1c, a TTTT spacer and the
sequence (F2) complementary to F2c. Backward inner primer (BIP) contains the
sequence (B1c) complementary to B1, a TTTT spacer and B2. The two outer
primers consist of B3 and the sequence (F3) complementary to F3c, respectively.
The mechanism and reaction steps of LAMP are illustrated in figure 1. Inner
primer hybridizes to F2c in the target DNA and initiates complementary strand
synthesis (Fig 1A). Outer primer F3, which is a few bases shorter and lower in
concentration than FIP, slowly hybridizes to F3c in the target DNA and initiates
strand displacement DNA synthesis, releasing a FIP-linked complementary strand,
which can form a looped out structure at one end (structure 4, Fig 1). The single
stranded DNA serves as template for BIP-initiated DNA synthesis and subsequent
B3-primed strand displacement DNA synthesis, leading to the production of a
dumb-bell from DNA (structure 6, Fig 1), which is quickly converted to a stem-loop
DNA by self-primed DNA synthesis (structure 7, Fig 1). This stem-loop DNA then
serves as the starting material for LAMP cycling, the second stage of the LAMP
reaction.

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Fakruddin 428

Figure 1: Schematic description of Loop Mediated Isothermal Amplification

(LAMP) assay (Notomi et al., 2000).
To initiate LAMP cycling, FIP hybridizes to the loop in the stem-loop DNA (structure
7, Fig 1) and primes strand displacement DNA synthesis, generating as an
intermediate one gapped stem-loop DNA with an additional inverted copy of the
target sequence in the stem and a loop formed at the opposite end via the BIP
sequence (structure 8, Fig 1). subsequent self-primed strand displacement DNA
synthesis yields one complementary structure of the original stem-loop DNA
(structure 10, Fig 1) and one gap repaired stem-loop DNA with a stem elongated
to twice as long (double copies of the target sequence) and a loop at the
opposite end (structure 9, Fig 1). Both these products then serve as template for a
BIP-primed strand displacement reaction in the subsequent cycles, a part of
which is designated the elongation and recycling step, illustrated in the right half
of figure 1. Thus, in LAMP the target sequence is amplified 3-fold every half cycle
(Notomi et al., 2000).
A typical reaction mixture of LAMP:
LAMP can be carried out in a total 25 µl reaction mixture containing 0.8 µM
each FIP and BIP, 0.2 µM each F3 and B3, 400 µM each dNTP, 1 M betaine, 20 mM
Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 4 mM MgSO4, 0.1% Triton X-100 and
the specified amounts of double-stranded target DNA. The mixture should be
heated at 95oC for 5 min, then chill on ice and 8U Bst DNA polymerase large
fragment should be added, followed by incubation at 65oC for 1 h and heating at
800C for 10 min to terminate the reaction (Notomi et al., 2000).

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LAMP - An Alternative to PCR 429

Factors affecting efficiency of LAMP:

Several factors affect the efficiency of LAMP reaction such as- 1.
Hybridization of the four primers to the target DNA in the initial step is critical for
the efficiency of LAMP. 2. The formation of stem-loop DNA from a dumb-bell
structure is critical for LAMP cycling. 3. The efficiency of LAMP also depends on
the size of the target DNA because one rate limiting step for amplification in this
method is strand displacement DNA synthesis. The size of the target DNA should
be set to less than 300 bp. 4. Selection of appropriate DNA polymerase is also
critical for LAMP efficiency. 5. Chemicals destabilizing the DNA helix have been
found to markedly elevate amplification efficiencies in LAMP (Notomi et al., 2000).
Design of primer
A set of two inner and two outer primers is required for LAMP. All four
primers are used in the initial steps of the reaction, but in the later cycling steps
only the inner primers are used for strand displacement synthesis. The outer
primers are known as F3 and B3 while the inner primers are forward inner primer
(FIB) and backward inner primer (BIP). Both FIP and BIP contains two distinct
sequences corresponding to the sense and antisense sequences of the target
DNA, one for priming in the first stage and the other for self-priming in later stages.
By using an additional set of two loop primers, forward loop primer (LF) and
backward loop primer (LB), the LAMP reaction time can be further reduced. The
size and sequence of the primers were chosen so that their melting temperature
(Tm) is between 60-65 °C, the optimal temperature for Bst polymerase. The F1c
and B1c Tm values should be a little higher than those of F2 and B2 to form the
looped out structure. The Tm values of the outer primers F3 and B3 have to be
lower than those of F2 and B2 to assure that the inner primers start synthesis earlier
than the outer primers. Additionally, the concentrations of the inner primers are
higher than the concentrations of the outer primers (Notomi et al. 2000).
Furthermore, it is critical for LAMP to form a stem-loop DNA from a dumb-
bell structure. Various sizes of loop between F2c and F1c and between B2c and
B1c were examined and best results are given when loops of 40 nucleotides (40nt)
or longer are used. The size of target DNA is an important factor that LAMP
efficiency depends on, because the rate limiting step for amplification is strand
displacement DNA synthesis. Various target sizes were tested and the best results
were obtained with 130-200 bp DNAs (Notomi et al. 2000).
Visualization of amplified product
Several methods can be used to detect positive LAMP reactions. The most
common is agarose gel electrophoresis, with the gel stained by an intercalating
agent such as ethidium bromide. Under UV illumination, the gel shows a ladder
like structure from the minimum length of target DNA up to the loading well,
which are the various length stem-loop products of the LAMP reaction.
Alternatively, given the large amount of LAMP product generated, products can
be directly visualised in the reaction tube after incorporation of SYBR Green I stain
which has high binding affinity to DNA (Karlesen et al. 1995).
Addition of a fluorescent detection reagent (FDR) to the LAMP reaction
mixture before starting the amplification allows the product to be directly
visualised under UV illumination and reduces contamination. Calcein in the FDR
combines initially with manganese ions and remains quenched. As
pyrophosphate ions are produced as a by-product of the LAMP reaction, they
bind with and remove manganese from the calcein, which results in detectable
fluorescence which indicates the presence of the target genes (Yoda et al.,
2007). Alternatively, a low molecular weight PEI can be added to the LAMP

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Fakruddin 430

product after centrifugation for 10s at 6000 rpm to form an insoluble PEI-product
complex containing the hybridized fluorescently labelled probe. Reaction tubes
can then be visualized with a conventional UV illuminator or by fluorescence
microscopy (Mori et al., 2006).
Another method for detection of positive LAMP reactions is to monitor the
increased turbidity in the reaction mixture in real-time with a turbidimeter. The
turbidity is derived from precipitation of magnesium pyrophosphate generated as
a by-product and this correlates with the amount of DNA amplified (Saleh et al.,
2008).
Recently, a colorimetric assay was developed that uses hydroxyl napthol
blue to detect the magnesium pyrophosphate by-product in successful LAMP
amplification (Goto et al., 2009). The hydroxyl naphol blue can be incorporated
into the LAMP reaction and the colour change visualized immediately after
amplification has been completed, and amplification can subsequently be
confirmed by agarose gel electrophoresis when necessary (Tomlinson et al.,
2010).
Advantages of LAMP
LAMP has many unique advantages for which it holds great promise to be
a method of choices for nucleic acid amplification. Advantages of LAMP are- 1.
LAMP uses simple cost effective reaction equipments (Parida et al. 2008). 2. Both
amplification and detection of nucleic acid sequences can be completed in
single step. 3. In addition the amplification efficiency of LAMP is very high 4. The
reaction proceeds rapidly as there is no need for initial heat denaturation of the
template DNA 5. Another important advantage of the isothermal amplification
techniques is their tolerance to some inhibitory materials such as a culture
medium and some biological substances that can affect the efficiency of PCR
(Kaneko et al. 2005). As LAMP is less affected by the various components of
clinical samples than PCR, there is no need for DNA purification (Nagamine et al.
2001). 6. LAMP is more specific, rapid and simple to perform than PCR. 7. As LAMP
method synthesizes a large amount of DNA, the products can be detected by
simple turbidity. 8. Expensive equipment is not necessary to give a high level of
precision, equivalent or greater, when compared to PCR techniques. 9. The
amplification efficiency of the LAMP method was extremely high because of its
no time loss for thermal change, based on its isothermal reaction. 10. The most
significant advantage of LAMP is its ability to amplify specific sequences of DNA
under isothermal conditions, thereby obviating the need for a thermal cycler. 11.
The approach is less costly 12. The amplification products can be visualized
directly. 13. This method has only one set of primers for the target DNA
amplification 14. LAMP seems less prone to the presence of irrelevant DNA than
PCR. 15. By combination with reverse transcription, LAMP can amplify RNA
sequences with high efficiency. 16. It is highly sensitive & able to detect DNA at as
few as 6 copies in the reaction mixture (Notomi et al., 2000)
Applications of LAMP
Considering the above mentioned advantages, LAMP is becoming a
valuable tool for the rapid diagnosis of infectious diseases in hospital laboratories
or for the rapid detection of pathogenic microbes in food (Yano et al. 2007). The
LAMP has been used for the detection of pathogens such as Mycobacterium
avium, Yersinia pseudotuberculosis, Mycoplasma pneumoniae and
Porphyromonas gingivalis (Enosawa et al. 2003; Saito et al. 2005; Yoshida et al.
2005). Multiple LAMP assays have been designed for microbial pathogen
detection (Hara-Kudo et al., 2007; Qiao et al., 2007; Yoneyama et al., 2007). It has
been successfully used to detect and amplify DNA viruses (Bista et al., 2007;
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LAMP - An Alternative to PCR 431

Enomoto et al., 2005; Iwata et al., 2006; Kaneko et al., 2005; Notomi et al., 2000;
Okomoto et al., 2004) and RNA viruses (Mori et al., 2006; Okafuji et al.,2005; Parida
et al.,2006; Parida et al.,2007; Thai et al., 2004). LAMP can be a simple and
valuable tool for the rapid diagnosis of infectious diseases by using very basic
facilities and should be easily applicable in the clinical laboratories of developing
countries.
Here, we summarized some of the most important applications-
(a) Detection of Pathogenic Bacteria
LAMP method has been used to detect various pathogenic
microorganisms with simple, rapid, and cost-effective features in recent years.
LAMP method was first used to detect stxA2 in Escherichia coli O157:H7 cells
(Maruyama et al., 2003). The mild permeabilization conditions and low isothermal
temperature used in the in situ LAMP method caused less cell damage than in situ
PCR. Higher-contrast images were obtained with this method than with in situ
PCR. Actinobacillus actinomycetemcomitans has been implicated in the etiology
of aggressive periodontitis, and the LAMP assay was used for the rapid detection
of A. actinomycetemcomitans in clinical specimens (Osawa et al., 2007). In
addition, Iwamoto et al., 2003 used the LAMP technology to specific detection of
Mycobacterium tuberculosis complex, Mycobacterium avium and
Mycobacterium intracellulure. LAMP has also been successfully used for specific
detection of pathogenic microorganisms such as Streptococcus pneumoniae
(Seki et al., 2005), Edwardsiella tarda (Savan et al., 2004), Edwardsiella ictaluri (Yeh
et al., 2005), methicillin-resistant Staphylococcus aureus (Misawa et al., 2007), and
Bacillus anthracis (Qiao et al., 2007).
(b) Detection of Fungi
The LAMP method has the advantage of speed and simplicity in detection
of fungi compared with the classic diagnostic methods such as the
histopathological test and biological material culture. For instance, Endo et al.,
2004 successively used LAMP method to succeed in detecting the presence of
the thermodependent dimorphic fungus Paracoccidioides brasiliensis.
(c) Detection of the Pathogenic Virus
LAMP method has been widely used in detection and diagnosis of DNA
and RNA virus. Up to now, there are many reports about LAMP method for
detecting DNA viruses. Fukuta et al., 2003 applied LAMP method to detect
tomato yellow leaf curl virus DNA. The detection sensitivity is 100 times higher than
the PCR. Qu et al., 2010 adopted LAMP method to diagnose porcine parvovirus
(PPV). Their results demonstrated that the LAMP assay could be well-applied to
laboratories, as a portable device and valuable tool for differential diagnosis of
PPV in the countryside. LAMP technology was also successfully applied to detect
the human herpes virus-7 (Yoshikawa et al., 2004), herpes simplex virus and
varicella-zoster virus (Kaneko et al., 2005), and human influenza A virus Hl-H3
(Poon et al., 2005). LAMP has also been applied successfully for RNA virus
detection with high efficiency by directly adding the reverse transcriptase to the
reaction mixture, which is termed the reverse transcription LAMP (RT-LAMP) (Chen
et al., 2009).
(d) Detection of Pathogenic Parasites
Protozoan parasites, common but important pathogens, are seriously
harmful to human, animals, and fish, which severely endanger human health and
the development of animal husbandry and aquaculture. LAMP-based technical
characteristics and its detection of protozoan parasites have also been widely
used, such as Trypanosoma (Kuboki et al., 2003), Plasmodium (Han et al., 2007),
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Fakruddin 432

Babesia (Ikadai et al., 2004), Cryptosporidium (Karanis et al., 2007), and

Tetracapsuloides bryosalmonae in aquatic animals (El-Matbouli & Soliman 2005).
(e) Detection of Mycoplasma
Mycoplasma infection can lead to a variety of infectious diseases.
Laboratory testing methods such as Mycoplasma culture, antigen detection, and
serological methods are generally adopted during clinical diagnosis of
Mycoplasma infection. These detection methods are time-consuming and
expensive. Therefore, Saito et al., 2005 developed a LAMP assay for the rapid
detection of Mycoplasma pneumoniae, and no cross-reactivity was observed for
other Mycoplasma species or respiratory bacterial species. It may be applied in
the routine diagnosis of M. pneumoniae infection in the clinical laboratory. And
Kawai et al., 2009 developed a LAMP method to detect Chlamydophila
pneumoniae infection. This assay exclusively amplified C. pneumoniae
sequences, and no cross-reactivity was observed for other Chlamydia species.
(f) Application in Tumor Detection
Tailor-made surgeries for patients with tumor have been under
consideration on the basis of the development of new approaches for minor
metastatic foci of malignant tumors. Accurate and reliable methods to detect
metastases in biopsy specimens with certain rapidity are essential for the
performance of these surgeries. Horibe et al., 2006 developed a rapid and
practical method to detect metastasis in specimens from patients with gastric
carcinoma using RT-LAMP to detect cytokeratin 19 (CK19) mRNA. The agreement
rate of CK19 expression detection by RT-LAMP and RT-PCR analysis was 31 out of
32 (97%). The RT-LAMP technique showed similar sensitivity to detect metastases
as nested RT-PCR assay.
(g) LAMP in SNP typing
LAMP-based SNP typing is an accurate, rapid and simple method that may
be useful especially for point-of-care testing. Because of the high specificity of the
LAMP method, only the target gene will be amplified from gene samples
containing homologous nucleotide sequences when using LAMP-based SNP
typing [Iwasaki et al., 2003; Nakamura et al., 2007]. Furthermore, because of the
characteristics of its amplification reaction, the LAMP method discriminates a
single nucleotide difference at each cycling step of the DNA replication, through
both ‘sense and antisense strand’ reactions, and the type of SNP can easily be
detected just by amplifying the DNA containing SNP in a single step. Due to the
simplicity and rapidity of the LAMP method, simple detection of SNP typing can
be achieved within 30 min. The products of the LAMP reaction, which was
performed in the presence of an intercalating dye, were detected within 30 min
without any post-reaction sample manipulation.
(h) Application in Detecting Genetically Modified Organisms (GMO)
Many detection methods have been developed and extensively used to
detect genetically modified organisms (GMOs) based on the LAMP reaction.
Cauliflower mosaic virus 35S promoter gene, a widespread genetic element, was
amplified by a set of LAMP primers (Fukuta et al., 2004). Lee et al., 2009 applied
the LAMP method to amplify GMO-related DNA sequences. Results showed that
detection of 0.01% GMO in equivalent background DNA was possible, and
dilutions of template suggested that detection from single copies of the template
may be possible using LAMP. Guan et al., 2010 reported one optimized visual
LAMP method for the detection of exogenous DNA targets from two GM soybean
events. This isothermal amplification could be performed within 40 min without
PCR equipment, and the derived LAMP products could be directly observed by
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LAMP - An Alternative to PCR 433

naked eye employing SYBR Green I dye instead of conventional gel

electrophoresis analysis. The limits of detection of these established visual LAMP
assays were about four copies of haploid soybean genomic DNA and which were
much higher than those of reported conventional PCR assays.
(i) Detection of ammonia-oxidizing bacteria
Aoi et al., 2006 used LAMP as a simple method for monitoring ammonia-
oxidizing bacteria. Their results showed that it was possible to quantify the initial
target DNA with sensitivity down to 102 copies while the background DNA from
non-targeted bacteria did not affect the quantitative capability of LAMP, which
suggested that the LAMP is effective for monitoring bacteria and their gene
expression in environment.
(j) Detection of Brettanomyces
Some species of Brettanomyces could cause turbidity or off-flavors in
wines, beer and soft drinks, resulting in significant economic losses worldwide.
Hayashi et al, 2007 established an identification method using LAMP which is
advantageous in terms of specificity, sensitivity, rapidity and simple operation
compared with PCR. They designed primer sets specially targeting the ITS region
of Brettanomyces (Dekkera anomala, Dekkera bruxellensis, Dekkera custersiana
and Brettanomyces naardenensis) which could detect 1×10 CFU/mL of
Brettanomyces yeast isolates from water, beer, wine and soft drinks.
(k) Detection of H5N1 avian influenza virus
RT-PCR with H5-specific primers recommended by WHO has been used
worldwide for H5-influenza virus detection following the outbreak of highly
pathogenic avian influenza (HPAI) caused by the H5N1 influenza virus in the end of
2003. However, this assay is time consuming and requires precision instruments
which prevent its being widely used, especially in some poor countries. Recently,
Imai et al. 2007 have used LAMP to develop a rapid laboratory diagnostic system
for HPAI and the sensitivity and specificity of the test have been validated. The
results suggested that the present LAMP system is a useful diagnostic tool for the
HPAI-H5N1 virus.
(l) Embryo sexing identification
Predetermination of embryo sex is very important for the embryonic
research. Detection of Y chromosome- specific sequences has widely been used
to predict the sex of offspring. Hirayama et al. (2006) have established an
efficient procedure for water buffalo embryo sex determination by identifying a Y
chromosome- specific sequence using LAMP, while a 12S rRNA was also amplified
by LAMP as a control reaction in both male and female. The results showed that
the minimal amount of the template DNA required appeared to be 0.1-10 pg,
and the total time for embryo sexing determination, including DNA extraction was
about 1 h.
(m) Quantitation of gene copy numbers by LAMP assay
Capitalizing on its exquisite sensitivity, LAMP has been designed to quantify
the amount of gene copies in a person's blood (load) thereby allowing physicians
to monitor their patients' disease progression and response to therapy. Assessment
of the load of the organisms/pathogens before, during and after therapy has
tremendous potential for improving the clinical management of diseases (Parida,
2008).
(n) Application of LAMP in aquaculture

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Fakruddin 434

Viral diseases in fish and shellfish can cause mass mortalities and the use of
specific pathogen-free stocks is an excellent way to prevent the introduction of
disease-causing agents into culture systems. A major problem lies in the
introduction of carriers from imported fish stocks, e.g. introduction of infectious
pancreatic necrosis virus/IHNV infected fish into Japan (Yamazaki 1974). Along
with robust legislation, a reliable and sensitive diagnostic technique is needed to
detect known and emerging pathogens. The development of diagnostic kits
based on nucleic acid detection is simpler and more cost-effective than
developing virus specific celllines.
Nucleic acid-based detection of protozoan and parasitic diseases in fish
for efficient management practice in culture systems has been progressing in
recent years. Many reports and reviews have been published on the detection of
parasites using molecular methods in fish and shellfish (Gasser & Monti 1997;
Cunningham 2002) including the use of ribosomal RNA genes and spacers by
PCR. However, cross-reaction with host tissues because of the presence of
complementing conserved rDNA sequences has been suggested as a cause of
false-positive results (Perkins & Martin 1999). Using LAMP the specificity could be
enhanced as the primers hybridize to six distinct sequences (El-Matbouli & Soliman
2005).
(o) LAMP in clinical diagnosis of virus infections
LAMP technology facilitates the detection of DNA or RNA of pathogenic
organisms and, as such, is the basis for a broad range of clinical diagnostic tests
for various infectious agents, including viruses and bacteria. These gene based
tests have several advantages over traditional antibody-based diagnostic
methods that measure the body's immune response to a pathogen. In particular,
LAMP is capable of detecting the presence of pathogenic agents earlier than
PCR even on day one of fever where the amount of gene copy number is
expected to be very low due to higher sensitivity with a detection limit of about 1-
2 copies (Parida, 2008). Both DNA and RNA viruses can be detected by LAMP and
diagnosis of several important emerging and re-emerging diseases by this
technique has already been reported. LAMP has been successful in detection of
viruses such as Human Papillomavirus type 6, 11, 16 and 18 (Hagiwara et al., 2007),
Varicella-zoster virus (Okamoto et al., 2004), Cytomegalovirus (Suzuki et al., 2006),
Severe acute respiratory syndrome (SARS) coronavirus (Hong et al., 2004), Dengue
viruses (1,2,3 & 4) (Parida et al., 2005), Newcastle disease virus (Pham et al., 2005),
Foot and mouth disease virus (Dukes et al., 2006), West nile virus (Parida et al.,
2004) etc.
Future prospects of LAMP:
LAMP method is a powerful innovative gene amplification technique
emerging as a simple rapid diagnostic tool for early detection and identification
of microbial diseases. Considering the advantages of rapid amplification, simple
operation, and easy detection, LAMP has potential applications for clinical
diagnosis as well as surveillance of infectious diseases in developing countries
without requiring sophisticated equipment or skilled personnel. During the last 10
years, this method has been successfully applied in molecular detection and
diagnostics for the detection of bacterial, viral, fungal, and parasitic diseases in
both animals and plants.
The integration of isothermal amplification and electrophoresis onto
microchips could lead to LAMP on Chips for quick and accurate identification of
disease producing genes at the patient’s bed side. LAMP has all the
characteristics required of real time assays (high sensitivity, quantitative) along
with simple operation for easy adaptability to field conditions. If these
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LAMP - An Alternative to PCR 435

characteristics of the LAMP method are used effectively, it should be possible to

develop simple genetic testing devices that have not been realized yet despite a
strong awareness of their necessity, in a wide range of fields, including infectious
disease testing, food inspection and environmental testing. This method will be
widely applied in clinical diagnostics, environmental monitoring, food safety, and
health fields with a broader development prospects in the future.
Conclusion
In conclusion, LAMP is a novel method of nucleotides amplification which can
amplify a few copies of DNA in less than an hour under isothermal condition and
with great specificity. There have been many reported researches which have
proven this method to be a successful molecular tool for the amplification of
target DNA sequence. The method provides us with a powerful biological tool to
be used in the research of clinical medicine, diagnosis of infectious diseases,
genetic disorders and genetic traits.
Acknowledgement
The author is indebted to Bangladesh Council of Scientific and Industrial
Research (BCSIR).
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